Lead Zirconate Titanate (PZT) sensors have become popular in structural health monitoring (SHM) using the electromechanical impedance (EMI) technique for damage identification. The vibrations generated during the casting process in concrete structures substantially impact the conductance signature’s  magnitude and sensitivity. The concept of smart sensing units (SSU) is presented, composed of a PZT patch, an adhesive layer, and a steel plate. It is embedded in the concrete structure to study the impact of damage since it has high sensitivity to detect any structural changes, resulting in a high electrical conductance signature. The conductance signatures are obtained from the EMI technique at the damage state in the 10–500 kHz high-frequency range. The wave propagation technique proposes implementing the novel embedded SSUs to detect damage in the host structure. The numerical simulation is carried out with COMSOL multiphysics, and the received voltage signal is compared between the damaged and undamaged concrete beam with the applied actuation signal. A five-cycle sine burst modulated by a Hanning window is employed as the transient excitation signal. For numerical investigation, six cases are explored to better understand how the wave travels when a structural discontinuity is accounted for. The changes in the received signal during actuator–receiver mode in the damage state of the host structure are quantified using time of flight (TOF). Furthermore, the numerical studies are carried out by combining the EMI-WP technique, which implies synchronous activation of EMI-based measurements and wave stimulation. The fundamental idea is to implement EMI-WP to improve the effectiveness of SSU patches in detecting both near-field and far-field damage in structures. One SSU is used as an EMI admittance sensor for local damage identification. Meanwhile, the same EMI admittance sensor is used to acquire elastic waves generated by another SSU to monitor damages outside the EMI admittance sensor’s sensing area. Finally, the experimental validation is carried out to verify the proposed methodology. The results show that combining both techniques is an effective SHM method for detecting damage in concrete structures. Structural health monitoring (SHM) using smart piezoelectric materials is used for real-time, continuous, or automated monitoring of structures. Non-destructive testing (NDT) techniques such as impedance meters, the ultrasonic-based monitoring approach , the ultIn recent years, there has been an increase in smart materials for structural monitoring. The electromechanical impedance (EMI) technique detects and localizes damage using the electromechanical coupling between the PZT patch and the host structure ,5. The PSeveral numerical and experimental studies have been conducted in concrete, steel, and aluminum structures to detect damage in the structures. Yang et al.  calculatThe PZT sensor, adhesive layer, and steel plate are combined to form the novel sensing unit. The stiffness and mass of the sensor are changed by varying the thicknesses of the adhesive layer and the steel plate, such that the peak frequencies vary due to the different stiffness ,16. SepeDamage detection via wave propagation is a transient dynamic phenomenon that aids in automated SHM ,24. The EMI and WP-based damage detection techniques have been widely used for SHM due to their sensitivity to small structural changes. Each of these techniques has its own technical merits, making them complementary. For example, the WP technique typically has a larger sensing range than the impedance technique, while the latter has better applicability to more complex structures. This paper aims to use EMI and wave propagation techniques to identify the structure’s damage precisely. The numerical investigations are carried out on the novel embedded SSUs to detect damage in the concrete structure. The analysis is carried out in COMSOL multiphysics , and theThe present work combines the two methodologies, implying synchronized EMI-based measurements and wave propagation activation. The basic idea is to use EMI-WP to improve the effectiveness of the SSU patch in detecting both near-field and far-field damage in structures. This is accomplished by detecting variation in the admittance spectrum of the undamaged and damaged concrete beam by (a) activating only wave propagation technology and (b) activating an EMI admittance sensor SSU-2 at the same time when a wave generator at SSU-1 is activated. The findings demonstrate that combining the two techniques is an effective way to identify damages in concrete structures.sZ) from a bonded PZT sensor by supplying the voltage at a certain frequency to it. EMI technique is utilized for monitoring the structural impedance response . The converse piezoelectric effect produces a mechanical vibration due to the input voltage pattern. The bonding interface will transmit mechanical vibration to the host structure, which further changes into electrical signatures by the direct piezoelectric action when it reaches another patch, which acts as a sensor. PZT transducers can be attached to the surface of a structure or embedded into a concrete structure at a predetermined spacing for effective use. The change in impedance governs the ratio of a received signal to actuating wave, enabling the location and extent of the damage to be determined. As a result, wave propagation mechanics can be used to evaluate and detect damage ,43,44,45c is the phase velocity in the axial direction, u is the displacement and x is the co-ordinate in the axial direction, E is Young’s modulus of elasticity, A is the area of bar element, and The second-order partial differential equation governing wave propagation through a prismatic bar element is expressed as:Different propagation modes and dispersion patterns determine the wave motion through the waveguide. There are three fundamental modes: longitudinal, flexural, and torsional modes. A numerical simulation is carried out to understand the wave propagation mechanics using COMSOL™ Multiphysics 5.5 on a concrete beam with 1000 mm × 100 mm × 100 mm dimensions. A numerical investigation is carried out to understand the impact of the damage through EMI response of the free-free PZT (before applying an epoxy coating) and embedded sensor in a concrete structure. These simulations are performed in COMSOL™ Multiphysics 5.5 using the piezoelectric effect under the module: structural mechanics, where a PZT-5A transducer is considered for the analysis. The material properties of PZT-5A are illustrated in S is the strain vector of size (6 × 1), T is the stress vector of size (6 × 1) in Pa, E is the electric field vector of size (3 × 1) in Volt/m, D is the electric displacement vector of size (3 × 1) in C/m2, d is the electric displacement per unit stress at a constant electric field of size (3 × 6) in C/N, and The constitutive relations of the PZT material according to the naming convention used in COMSOL™ Multiphysics :(15)S=TCFor numerical analysis, the three-dimensional (3D) modeling of the PZT sensor of the dimension 10 mm × 10 mm × 1 mm is considered where one of the 10 mm faces of the PZT sensor is used as a terminal with 1V applied voltage, while the opposite face is used as a ground as shown in In this section, the novel smart-sensing units (SSUs) are kept inside the concrete structures to examine the structural damage as they show high sensitivity to possible changes in the host structure. The SSU consists of a PZT sensor, adhesive layer, and steel plate, as shown in When two or more PZT transducers are considered in the analysis, one of the PZT patches acts as an actuator, i.e., excited by a transient voltage  and the other acting as a receiver utilizing direct piezoelectric effect. PZTs are attached to the surface of a structure or embedded in the concrete structure at a predetermined spacing. A schematic diagram of wave propagation (WP) with a PZT transducer is shown in A concrete beam of dimension 1000 mm × 100 mm × 100 mm and a PZT patch of dimension 10 mm × 10 mm × 1 mm is considered for the analysis. The material properties of considered PZT-5A are illustrated in Undamaged concrete beam with surface-mounted PZT patches;Damaged concrete beam with surface-mounted PZT patches;Undamaged concrete beam with surface-mounted SSUs;Damaged concrete beam with surface-mounted SSUs;Undamaged concrete beam with embedded SSUs;Damaged concrete beam with embedded SSUs.In this section, the numerical investigations are performed considering six different cases, which are discussed below:FE simulation is performed for all the above cases. The discretization of the selected FE models is carefully allocated to assure the convergence to the proper solutions. Therefore, the considered FE models meet the commonly enforced accuracy standard of having at least seven FEs for the shortest wavelength inside the signal bandwidth of any waves that may travel in the waveguide :(18)Δx ≤−8. First, the surface-mounted PZT is considered without any damage in the beam to obtain the response spectrum in the actuator–receiver mode. Damage is introduced in the beam as structural discontinuity, and WP response is recorded in the time domain. Changes in the stiffness of certain elements in the mesh are used to introduce damages in this study. In this case, a surface-mounted SSU is considered to obtain the response spectrum in the actuator–receiver mode. The dimension considered for the concrete beam and the SSU is similar to the previous analysis. The material properties of the steel block are illustrated in Damage of 20 mm depth and 10 mm thickness is introduced at a location 300 mm away from SSU-1, and WP responses are recorded in the time domain. WP measurement is performed on the embedded SSU in the concrete beam to obtain the time-dependent transmitted wave. In this analysis, one SSU is excited by a transient voltage acting as an actuator. The converse piezoelectric effect will produce a mechanical vibration that follows the voltage input pattern in the form of a five-cycle sine burst modulated by a Hanning window. The bonding interface will transmit mechanical vibration to the host structure, which further changes into electrical signatures by the direct piezoelectric action when it reaches another patch, which acts as a sensor. In this case, a structural discontinuity is introduced in the beam to study the changes occurring in transmitting waves, where damage is introduced as a crack with a depth of 20 mm and a thickness of 10 mm. The presence of damage along the direction of wave passage causes a change in the amplitude of the wave traveling. ith value of reference signature, and ith value of the compared signature. damaged concrete beam with surface-mounted PZT patches) and case-4 (damaged concrete beam with surface-mounted SSUs), using the respective undamaged cases as a reference. It is feasible to conclude that utilizing WP response with SSU improves the RMSD value for damage detection, indicating that SSUs are more effective than smart PZT sensors. Similarly, the RMSD plot is obtained for case-4 (damaged concrete beam with surface-mounted SSUs) and case-6 (damaged concrete beam with embedded SSUs) with case-2 (damaged concrete beam with surface-mounted PZT patches) as reference data. The statistical metric to quantify the changes in the waveform is employed using root mean square deviation (RMSD) expressed as:The performance and reliability of damage detection are enhanced using impedance and guided wave signals simultaneously obtained from PZTs attached to a target structure . The proA numerical investigation is carried out in a concrete beam with a rectangular cross-section of 100 mm × 100 mm and a length of 1000 mm, as shown in (A)Active wave propagation in an undamaged concrete beam;(B)Active wave propagation in damaged concrete beam;(C)EMI admittance sensor SSU-2 is activated simultaneously with a wave generator at SSU-1 in an undamaged concrete beam;(D)EMI admittance sensor SSU-2 is activated simultaneously with a wave generator at SSU-1 in a damaged concrete beam.Four different conditions are investigated:The comparison of the Fast Fourier Transform (FFT) admittance spectrum for an undamaged and damaged concrete beam with active wave propagation is shown in From the plot, it is observed that the sensing capabilities of SSUs are enhanced when EMI-WP techniques are simultaneously enabled, which helps in better damage detection. The RMSD index of the absolute value of the admittance spectrum calculated under the investigated four distinct conditions is shown in The experimental study is performed on the concrete beam with embedded PZT patches to detect damage using wave propagation technology . The tesFor wave generated by piezoelectric transducers, in the transient analysis, a 3.5 cycle tone burst modulated by a Hanning window is given as actuating signal, expressed as:Likewise, for a damaged concrete beam for different crack depths and hole diameters with embedded SSUs, the RMSD value increases. The figure shows that using embedded SSUs for damage detection in concrete structures may effectively monitor damage through WP response, making embedded SSUs a more effective modeling technique for damage identification than existing smart aggregate models. Combining the EMI-WP technique implies synchronized activation of EMI-based measurements and wave propagation in the proposed technique for both near-field and far-field damage detection. A numerical investigation is carried out in a concrete beam of dimension 406.4 mm × 127 mm × 127 mm with two embedded SSUs as smart aggregates towards the two ends of each specimen on the central axis, as shown in Four different conditions are investigated, as explained in The sensing capabilities of SSUs are improved when EMI-WP techniques are combined, and the RMSD value is increased, resulting in greater damage identification. As a result, the embedded SSU is recommended as a more effective modeling technique for damage identification than the existing smart aggregate models.WP-based measurement shows that damage in the structure is successfully detected utilizing the embedded SSU by observing the variation in magnitude of the received voltage signal and improved RMSD value.Comparing the WP response in the actuator-sensor mode of the embedded SSU, it is observed that the decrease in the magnitude of the received voltage signal is because of a crack in the transmission path, and the increase in the time of flight is due to the presence of crack along the path of the wave propagating.The numerical value of the RMSD employing an SSU instead of PZT patches for damage detection exhibited by six separate cases in the wave propagation technique showed that steel–PZT models are preferable to smart material PZT-based models.EMI measurements are particularly important because they can detect damage to the embedded PZT patch or debond between the epoxy and concrete interface. Therefore, combining EMI and WP measurements for damage identification is useful for detecting relatively close and far damages in the structures.The results of the combined EMI-WP approach using four structural conditions show that the presence of damage is indicated by a change in the FFT admittance spectrum, as well as by the increment in the RMSD value from a damaged concrete beam where only wave propagation is active to EMI admittance sensor SSU-2 being activated simultaneously.The numerical results are compared with experimental analysis for different crack lengths and holes, which shows a good fit between the 3D FE model results and experimental results.The FE model results are compared with the experimentally available smart aggregate and proposed embedded SSU for damage detection, displaying enhanced damage index value. Thus, it is recommended that the embedded SSU results in a more effective modeling technique than the existing smart aggregate models for damage detection.The combined EMI-WP-based robust damage detection is developed by utilizing impedance and wave signals simultaneously obtained from SSUs to enhance the performance and reliability of damage diagnosis.This paper explains how to use EMI and WP to identify damage in the concrete structure. The WP technique is utilized since EMI measurements cannot identify damage outside the PZT sensor’s detection range. The following conclusions are derived from the study:


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.Neutrophils are the first line of defence against invading pathogens. Although neutrophils are well-known professional killers, some pathogens including  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.The leishmaniases are a complex of diseases caused by  HGF/c-MET signalling regulates numerous biological pathways such as proliferation, survival, migration and tissue regeneration . AberranIn addition to the role of neutrophils in cancer, neutrophils are also the first line of defence against invading pathogens. However, in addition to their well-characterized killing functions, neutrophils can also play deleterious roles in several infections including some forms of leishmaniasis ,9.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 [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 [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 [ote form ,11. L. mresponse –14. We pe burden .L. major was shown to enter neutrophils and persist in these cells, a process referred to as the Trojan horse model [L. mexicana infection [Leishmania in neutrophils is thought to promote subsequent parasite dissemination into the host.se model ,16. Persnfection ,17. TrapLeishmania mexicana. We report here a prominent role for c-MET expression on neutrophils in the control of Leishmania-induced cutaneous pathology.Here, we aimed to investigate the role of c-MET activation in neutrophils in the context of parasite infection, more specifically following infection with 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 . 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 . 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 . Similarly, L. mexicana promastigotes induced only a slight increase in c-MET expression in bone marrow-derived dendritic cells (BMDCs) . 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 . Very low mRNA induction was observed in BMMs and BMDCs exposed to L. mexicana amastigotes . 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 , showing that the upregulation of c-MET on neutrophils by Leishmania mexicana extends to other Leishmania spp.In order to investigate the role of c-MET in neutrophils during L. mexicana-DsRed at MOI of 5 and c-MET expression was assessed by flow cytometry. Non-infected (unexposed) neutrophils were used as a control. A significant increase of c-MET expression was observed in infected neutrophils (DsRed+) compared to non-infected cells (DsRed-), as shown in the representative flow cytometry plot ( and the corresponding mean fluorescent intensity (MFI) . We further show that parasites need to be alive to induce c-MET expression in neutrophils, as demonstrated by the lack of c-MET induction observed in response to dead Leishmania parasites . To investigate if Leishmania triggering of TLR receptors is involved in c-MET induction on neutrophils, we exposed MyD88-/-, TLR4-/- and WT neutrophils to L. mexicana and analyzed the levels of c-MET expression by flow cytometry. No difference in c-MET induction was observed in all the conditions tested, showing that L. mexicana-dependent c-MET induction in neutrophils is TLR-independent . To assess if c-MET induction depended on nuclear factor (NF)-κΒ activation, BMNs were treated or not with the highly selective IκΒ (IKK) inhibitor, which is critical for the activation of NF-κΒ [L. mexicana. Impairment of NF-κΒ activation resulted in partially decreased L. mexicana-induced c-MET levels . These data show that c-MET induction by L. mexicana is TLR independent and involves NF-κB activation.To further explore the impact of infection on c-MET expression, we determined whether parasite internalisation induced c-MET surface expression in neutrophils harboring or not parasites. First, BMNs were exposed for 16h to fluorescent of NF-κΒ , and infL. mexicana infected BMMs. Consistent with our mRNA data, infected and non-infected macrophages expressed low levels of c-MET , and we did not observe any change in c-MET expression between non-infected and infected BMDCs . Collectively, these data indicate that in vitro, c-MET expression is upregulated specifically in neutrophils that have phagocytosed the parasites suggesting that the parasite itself is inducing c-MET expression.We also assessed c-MET surface expression in in vivo at the site of infection, during the chronic phase of L. mexicana disease. C57BL/6 mice were intradermally (i.d.) infected with L. mexicana metacyclic promastigotes in the ear. Ten weeks after the infection, infected ears were collected, processed and c-MET expression in neutrophils, monocytes and dendritic cells was analysed by flow cytometry. The highest level of c-MET was observed in neutrophils, whereas monocytes and dendritic cells showed significantly lower expression. . The levels of c-MET were also assessed in situ, by immunofluorescence in cryosections of the infected ear 7 weeks after L. mexicana infection. c-MET was expressed in the epidermis as well as in the dermis including expression in neutrophils , Colocalized c-MET with neutrophils is shown in a three-dimensional representation . Around 30% of neutrophils were expressing c-MET as quantified by Imaris . Moreover, not only was the protein present, but the c-MET pathway was shown to also be active in these cells, as observed by immunofluorescence staining of phospho-c-MET (P-c-MET) in Ly6G+ cells . Colocalized P-c-MET with neutrophils is shown in a three-dimensional representation . Similarly, we were able to detect activation of this pathway in MR+ dermal macrophages . These data demonstrate the activity of this kinase in cutaneous Leishmania lesions.We then analysed c-MET expression L. mexicana metacyclic promastigotes. Three weeks post-infection with 104 metacyclic L. mexicana, when the first wave of neutrophil is starting at the site of infection with the onset of a lesion (mostly redness) and seven weeks post infection, when the cutaneous lesion is well established, the presence of HGF was analysed by ELISA in ear lysates. A significant increase in HGF levels was observed in L. mexicana infected ears compared to the levels observed in the ears of non-infected naive mice . In contrast, no significant difference in the levels of serum HGF was observed between uninfected and infected mice  indicating a local, rather than a systemic regulation of c-MET during infection.We then assessed if the level of HGF, the only known ligand for c-MET, was regulated at the site of infection. To this end, C57BL/6 mice were injected i.d. with L. mexicana-induced neutrophil effector functions, BMNs derived from mice genetically deficient for c-MET in neutrophils  and control littermate mice  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  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 . 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 . To assess if the absence of c-MET would have an impact on the parasite number per neutrophils, BMNs from WT  and c-MET deficient  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 , confirming our results obtained by conventional flow cytometry.c-MET expression was previously shown to impact neutrophil function in the tumor environment . To assefl/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 . 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 , 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.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;Metin 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 106L. 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 [+CD11b+Ly6G+ neutrophils was analyzed using the DHR123 probe. No difference was observed between c-MET deficient and WT neutrophils . 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 . 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.We then measured L. mexicana lesion contributes to the cutaneous pathology [fl/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 . 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 . Ten weeks post-infection, the number of CD11b+ cells recruited in the infected ear was comparable between the two groups of mice  but the frequency of infected (DsRed+) cells in the CD11b+ population was significantly reduced in Mrp8;Metfl/fl infected ears . Accordingly, neutrophils, monocytes and dendritic cells were less infected in Mrp8;Metfl/fl compared to control Mrp8;Metwt/wt mice . 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 .Early neutrophils infiltration in athology . Thus, wFig 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) . 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 .The ear cellularity at the infected site was analysed by flow cytometry (+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 . 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 . Correspondingly, the frequency of CD4+IL-4+ T cells was lower in the ears of Mrp8;Metfl/fl compared to control group . Collectively, our results indicate that c-MET signalling in neutrophils is contributing to the development of the pathology associated with L. mexicana infection.To analyse the type of adaptive immune response developing in these mice, we measured the frequency of CD4L. mexicana metacyclic promastigotes and twenty-one days post infection mice received the c-MET inhibitor capmatinib intraperitoneally twice a day (b.i.d.) for 20 consecutive days. Control mice received the vehicle solvent used to dilute the capmatinib as illustrated . Already 4 days post treatment, we observed a decrease in lesion size in mice receiving capmatinib injection compared to control mice, that became statistically significant 15 days post initiation of the treatment and remains so until the end of the treatment. . The parasite load present in the infected ears at the end of the treatment, as analysed by limiting dilution assay (LDA), showed comparable parasite burden between the two groups . Immunofluorescence of ear cryosection confirmed the success of the treatment, as the phospho c-MET signal observed in the vehicle group was almost abolished in the capmatinib treated group . Furthermore, a significant decrease in the Ly6G+ area at the site of infection was observed in mice treated with the c-MET inhibitor, correlating with the results obtained with mice genetically deficient in c-MET in their neutrophils. Interestingly, the area covered by macrophages was similar between the two groups of mice, indicating that the systemic treatment affected specifically the presence of neutrophils at the lesion site . To further quantify the infiltration of immune cells upon c-MET inhibition, we assessed the frequency of recruited cells at the site of infection by flow cytometry. We found an equal number of CD11b+ cells in mice treated or not with the c-MET inhibitor . In line with the immunohistology data, we detected a significant decrease in neutrophil infiltration in the ears of capmatinib-treated mice . In contrast, the frequency of monocytes and dendritic cells was similar in the two groups .Patients seek treatment when they harbour a defined cutaneous lesion. To translate our results to potential clinically relevant settings, c-MET signalling was systemically blocked once a cutaneous lesion was established in mice. To this end, C57BL/6 mice were infected i.d. with + T cells expressing IFN-γ and IL-4 at the site of infection and in dLN was analysed by flow cytometry. No difference in the frequency of IFN-γ+ and IL-4+ CD4+ T cells was observed in the two groups of mice in the dLN . However, at the site of infection a significant increase in the levels of intracellular IFN-γ was detected in CD4+ T cells of capmatinib-treated mice compared to control mice . No difference in the low frequency of CD4+IL-4+ T cells was observed in the infected ears. Overall, these results indicate that blocking c-MET once a lesion is established, impairs further recruitment of neutrophils, leading to a decrease in lesion size and modifications in local IFN-γ levels without impacting the parasite burden.We then analysed if the adaptive immune response was modified at the end of treatment in capmatinib-treated mice. To this end, the frequency of CD4L. mexicana infection in vitro, neutrophils upregulated c-MET despite the lack of detectable HGF levels in the culture supernatant. Furthermore, c-MET expression was significantly more induced in infected versus non-infected bystander neutrophils. In contrast, infection of DCs and macrophages did not induce significantly c-MET expression in vitro. These results demonstrate that the parasite itself induces c-MET expression in neutrophils. Interestingly, this induction was also observed in other Leishmania spp such as L. major, suggesting that Leishmania-induced c-Met expression contributes to neutrophil recruitment in leishmaniasis. Other pathogens including bacteria, virus and other parasites were also shown to promote c-MET activation in target cells [Plasmodium sporozoites in hepatocytes [Listeria monocytogenes was reported to bind to the extracellular region of c-MET expressed on epithelial cells, promoting bacterial internalization [Salmonella (S.) typhi express the virulence factor S. typhi invasin (STIV), which directly binds c-MET, favouring epithelial cell invasion [c-MET expression in neutrophils has been shown to be critical for their migration during inflammatory settings. Furthermore, c-MET expression on neutrophils contributes to the killing of tumour cells in the tumour microenvironment ,19,20. Tet cells –30. In aatocytes ,31. In blization ,32. SimiLeishmania are recognized by several TLRs such as TLR2, 4, 7 and 9 [L. mexicana. However, induction of c-MET was not linked to TLR4 or other TLR triggering by L. mexicana, as MyD88-/-, TLR4-/- and WT BMNs exposed to L. mexicana were equally capable to induce c-MET surface expression. We further show that activation of the NF-κΒ pathway contributes to the induction of c-MET on neutrophils by L. mexicana. These data are in line with a previous report showing that LPS- and TNF- mediated c-MET induction required NF-κΒ activation[It has been previously shown in cancer that c-MET expression in neutrophils is regulated by TNFα-dependent NF-κB activation . c-MET e 7 and 9 –41, suggPlasmodium, the causative agent of malaria, was reported to induce HGF secretion, the activation of c-MET by hepatocytes and invasion of these cells [Plasmodium spp did not [L. mexicana induced the expression of c-MET on neutrophils but did not show any impact on the phagocytosis of the parasites. The production of radical oxygen species (ROS) by neutrophils is one of the major effector mechanisms of neutrophil killing. Infection of Mrp8;Metfl/fl c-MET-deficient neutrophils resulted in increased ROS release compared to their wild type counterpart. This led to a small but statistically significant decrease in live parasites released by in vitro infected neutrophils. These data suggest that c-MET activation on neutrophils decreases ROS production by infected neutrophils. However, in vivo, following infection with a high dose of parasites, the impact of c-MET expression on ROS production by neutrophils is compensated by other local activating factors of neutrophils. Similarly, no difference in NO production was observed 24 hours after infection.Cell traversal (hepatocytes) observed in several species of se cells , while o did not . L. mexiLeishmania inoculation [L. mexicana-induced c-MET activity selectively in neutrophils, we infected mice with a low dose of parasites, a process inducing a parasite-dependent neutrophil recruitment around three weeks post infection, at a time when the multiple neutrophil-attracting factors induced by the physical damage to the ear do not persist. We showed that the onset of parasite-induced neutrophil recruitment observed from three weeks post infection correlated with increased HGF levels at the site of infection. In addition, Mrp8;Metfl/fl mice infected with a low dose of parasites controlled better their lesion development and harboured decreased parasite burden compared to control mice similarly infected. Importantly, impaired neutrophil recruitment was observed already early in infection, at a time when neutrophils start to infiltrate the site of infection in response to parasites, but when the ear inflammation is barely detectable and no difference in lesion score is apparent between Mrp8;Metfl/fl and Mrp8;Metwt/wt ears. These data demonstrate that the absence of c-MET in neutrophils contributes to the reduction in neutrophil recruitment observed throughout the infection.Numerous factors, including the dose of parasites injected and the inflammation caused by the local tissue damage are involved in the first wave of neutrophil recruitment following i.d. culation ,40,43,44+IFNγ+ T cells was observed at the site of infection both in mice genetically deficient for c-MET in neutrophils and in the lesion of mice treated with the c-MET inhibitor capmatinib. These results suggest a suppressive role of c-MET in the effector function of CD4+IFNγ+ T cells at the site of infection. An effect of c-MET expression on neutrophils on the T cell effector functions was previously reported for CD8+ T cells in an experimental model of cancer, where the fitness of adoptively transferred CD8+ T cells in Mrp8;Metfl/fl mice was enhanced [+IFNγ+ T cells and the decrease in CD4+IL-4+ T cells observed at the site of infection correlated with decreased parasite burden in the lesion of Mrp8;Metfl/fl mice. Collectively, our data suggest that c-MET expression on neutrophils also contributes to the impairment of the local adaptive immune response.An increase in the frequency of CD4enhanced . The incfl/fl mice and mice treated with the c-MET inhibitor capmatinib, is that pharmacological inhibition of c-MET acts on all cells, while in Mrp8;Metfl/fl mice, impaired c-MET expression is restricted to neutrophils. We observed induced expression of phospho-c-MET in dermal macrophages at the lesion site, showing c-MET activation in dermal macrophages. Inhibition of c-MET in hematopoietic cells was previously shown to decrease total NO levels at the tumour site, a process correlating with decreased iNOS by neutrophils [L. mexicana-induced NO production in neutrophils but the global pharmacological inhibition of c-MET in vivo could impair either directly or indirectly the IFNγ-induced NO production by macrophages, a process critical in killing the parasites.Despite increased levels of IFNγ, decreased parasite burden was not observed in mice following systemic inhibition of c-MET signalling once a cutaneous lesion is established, this could be linked to the length of the treatment. A major difference between Mrp8;Mettrophils . We did L. mexicana infection. This favours their migration to the site of infection, providing a protective niche for the parasites that can further expand in the host, contributing to the chronicity of the disease. Furthermore, we showed that c-MET activation on neutrophils exacerbates the cutaneous pathology, prompting us to treat the cutaneous lesion with a c-Met inhibitor, with significant impact on the pathology. Collectively, our data reveal a potential use for c-MET inhibitors in the control of the cutaneous Leishmania lesion.In summary, our results reveal that c-MET expression is activated in neutrophils during Animal experimentation protocols were approved by the veterinary office of the Canton of Vaud (Authorization 1266.6–7 to F.T.C.) and were done in accordance to cantonal and federal law as well as the principles of the declaration of Basel.fl/fl (Cre-positive) and Mrp8;Metwt/wt (Cre-negative) on C57BL/6 background were provided by Professor Massimiliano Mazzone (University of Leuven) [-/- and TLR4-/- mice on C57BL/6 background were obtained from Prof. Shizuo Akira (University of Osaka). Mice were bred in pathogen-free conditions at the Epalinges centre. 6–8 weeks-old female mice were used in the experiments.C57BL/6 mice were purchased from Envigo . Mrp8;MetL. mexicana (MYNC/BZ/62/M379) WT, L. mexicana-DsRed [L. major LV39 (MRHO/SU/59/P) [in vivo infection metacyclic promastigotes were needle injected in the ear dermis, lesion development was measured weekly using a caliper as previously described [na-DsRed  and L. mSU/59/P)  were useSU/59/P) . AmastigSU/59/P) . Brieflyescribed . A scoreL. mexicana parasites were obtained after submitting promastigotes to seven freeze-thaw cycles.Dead in vivo. Mice were given 5 mg/kg of capmatinib intraperitoneal (i.p.) twice a day (b.i.d) during the indicated time as previously described [c-MET inhibitor capmatinib  was used to block c-MET escribed . ControlEars were collected, fixed in 4% PFA at 4°C overnight and washed. Then, they were cryoprotected in 30% and embedded in Tissue-Tek OCT (Sakura). 20- or 8-μm thick OCT sections were thawed for 30 minutes at room temperature, fixed in 4% PFA for 10 minutes and permeabilized with 0.3% Triton X-100 in PBS. After 30 minutes in blocking buffer , slides were incubated with primary antibodies overnight at 4°C. The next day slides were washed with 0.3% Triton X-100 in PBS for 30 minutes and appropriate alexa secondary antibodies 488/555 were added 1:500 in blocking buffer for 1 hour at RT. After that, slides were washed with 0.3% Triton X-100 in PBS for 30 minutes and mounted.+ and F4/80+ cells was performed using ImageJ software. 3D rendering and visualisation were also generated using the Imaris cell imaging software.The following antibodies were used: either unlabelled or biotin-labelled Ly6G (Biolegend), anti-F4/80-APC (Invitrogen), anti-MET (Abcam), anti-Phospho-MET (Tyr1234/1235) , anti-CD206 (Biolegend). Images were acquired on a Zeiss LSM880 and analysed using Imaris. Quantification of Ly6GMyeloid cells were isolated from the bone marrow (BM) of naïve female mice. Femora and tibia were collected and BM was obtained by flushing the bones with RPMI media. Neutrophils were purified by positive magnetic-activated cells sorting (MACS) using anti-Ly6G MicroBeads UltraPure isolation kit, according to manufacturer’s instructions (Miltenyi Biotec). The purity of neutrophils (>95%) was assessed by cytospin assay or flow cytometry. BM progenitor cells were differentiated for 6 days in 20% L929-MCSF supernatant to generate BM-derived macrophages. BM progenitor cells were differentiated for 7 days in 20% AO2-GMCSF, in order to obtain BM-derived dendritic cells.E. coli O127:B8 (Sigma-Aldrich). For NF-κΒ inhibition, neutrophils were pre-treated for 1h at 37°C with 10μM of IKK inhibitor III (BMS-345541) .When indicated, cells were treated with 50 ng/mL of LPS from Myeloid cells were lysed in RLT buffer, and mRNA was extracted using the RNeasy Plus Mini kit (Qiagen) according to manufacturer’s instructions. 500 ng of mRNA was reverse transcribed into cDNA using random nonamers primers (Microsynth) and M-MLV RT polymerase (Promega). cDNA was then purified using the QIAquick PCR purification kit (Qiagen) according to manufacturer’s instructions. c-MET gene expression was assessed by Real-time PCR using the SYBR green kit (Roche) and the LightCycler system (Roche). c-MET expression was normalized to the housekeeping gene hypoxanthine phosphoribosyl transferase (HPRT), using the comparative threshold cycle method for relative quantification. The following primers (Microsynth) were used: c-MET Forward: 5’-CTGCTCTGGAGGACAAGACC-3’, Backward: 5’- GAGTTGATCACATGCCAAGC-3’; HPRT Forward: 5’- GTTGGATATGCCCTTGAC-3’, Backward 5’-AGGACTAGAACACCTGCT-3’.Ears were collected at indicated time points and processed to obtain a single cell suspension as previously described . BrieflyStained cells were analyzed using LSR-Fortessa (BD Bioscience) and analysed with FlowJo software (Tree Star). The following antibodies were used to detect cell surface expression: anti-CD45-PerCPCy5.5, anti-CD11c-PeCy7, anti-Ly6C-FITC, anti-Ly6G-PE and anti-Ly6C-APC (BD Bioscience); anti-Ly6G-APC/Cy7 and anti-CD4-AF700 (Biolegend); anti-CD8-APC, anti-CD11b-PB, anti-IFNγ-PeCy7 and c-MET-FITC (Invitrogen); anti-IFNγ-PE, anti-IL-4-FITC (eBioscience) and anti-Annexin V-PeCy7 (eBioscience).ex vivo experiments, whereas DAPI (Sigma-Aldrich) was used for in vitro experiments. Parasite-infected cells were identifies based on the DsRed fluorescence of transgenic parasites.Cell viability was analyzed using the Live/Dead fixable Aqua Dead Cell Stain Kit (Invitrogen) for Samples were analysed using an Image Stream cytometer  at low speed and 60x magnification. Debris and free parasites were excluded from the acquisition based on the area and aspect ratio of the bright field. Internalized parasites were differentiated from non-internalized parasites according to the internalization of the bright parasite spots within the membrane marker mask. The number of parasites per neutrophil was determined using the spot count within the parasite channel feature. The IDEA software was used for the analysis. Neutrophils were stained with Ly6G-APC/Cy7 (Biolegend) antibody and cell viability was assessed using DAPI (Sigma-Aldrich).L. mexicana. 20 μg/ml of luminol (Carbosynth) was added to the cells and ROS chemiluminescence was measured over time at all wavelengths using a plate reader . To measure ROS and NO by flow cytometry, ear cells were incubated with 1.2 μM DHR123 molecular probe (Thermo Fisher Scientific) or 1 μM DAF-FM probe (Invitrogen), respectively for 45 min at 37°C.To measure ROS formation by luminol based chemiluminescence assay, bone marrow neutrophils were incubated in X-vivo (Lonza) medium in a white opaque 96-well plate  and exposed to 6 dLN cells or 105 ear cells were stimulated for 4h at 37°C with 50 μg/mL PMA, 500 μg/mL Ionomycin and 1 μg/mL GolgiPlug (BD Bioscience). Cells were stained for extracellular markers and Live/Dead dye, and fixed with 2% PFA. Saponin was used to permeabilize the cells. Intracellular cytokines were stained for 45 min by directly diluting the antibodies in saponin.Intracellular cytokines were detected by flow cytometry in ears and dLNs. 10Ears were collected, frozen in liquid nitrogen and homogenized in RIPA lysis buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Roche) using a magnetic bead and a tissue lyser (Qiagen). The ear lysate was analysed for HGF by ELISA (R&D systems) according to manufacturer’s instructions.In order to estimate the number of living parasites, single cell suspensions from infected mice were serially diluted in supplemented M199 media. Each dilution was loaded in 8-fold replicates, in a 96-well plate containing 100μl of rabbit blood agar. The plates were cultured at 26°C during 7 days. The number of wells positive for parasites was determined by microscopy, whereas the number of parasites was determined using the ESTIMFRE software as previously described .P values were determined using GraphPad Prism version 8. Statistical difference between two groups was analysed using Mann-Whitney nonparametric t-test. Whereas difference between multiple groups were determined using one way analysis of variance (ANOVA).S1 FigA) BM-derived neutrophils (BMNs) were isolated and exposed for 16h to L. mexicana metacyclic promastigotes at the indicated multiplicity of infection (MOI). The apoptotic status of control and L. mexicana-exposed neutrophils was analyzed by flow cytometry using Annexin V and DAPI staining. The frequency of live (AnV-DAPI-), early-apoptotic (AnV+DAPI-) and late apoptotic (AnV+DAPI+) neutrophils is shown. B) BMNs were isolated from Mrp8:Metfl/fl and control Mrp8;Metwt/wt littermates and exposed to L. mexicana-DsRed for 2 or 20h at indicated MOI. The frequency of live (DAPI-) BMNs was assessed by flow cytometry. Data are representative of 3 independent experiments with n≥ 2.(TIF)Click here for additional data file.S2 FigA) BM neutrophils (BMNs) were isolated and co-cultured in vitro with metacyclic L. major LV39 for 16h at a MOI of 5. c-MET mRNA levels were analysed by RT-qPCR. Unexposed BMNs were used as negative control and LPS treated cells as positive control. B) BM-derived macrophages (BMMs) and C) BM-derived dendritic cells (BMDCs) were similarly infected and c-MET expression was assessed by RT-qPCR. Data are normalized to endogenous levels of HPRT mRNA and expressed as fold increase relative to expression levels measured in control cells. Data are representative of ≥ 3 experiments, n = 3/group.(TIF)Click here for additional data file.S3 FigA) BMNs were isolated from MyD88-/- and C57BL/6 mice and co-cultured for 16h with L. mexicana metacyclic promastigotes at a MOI of 5. c-MET expression in infected or non-infected (unexposed) neutrophils was assessed by flow cytometry. The MFI of a representative experiment is shown. B) BMNs isolated from TLR4-/- and C57BL/6 mice were processed as indicated in A. The MFI of a representative experiment is shown. C) BMN were isolated from C57BL/6 mice and pre-treated for 1h with the IKK inhibitor III. Cells were then exposed to L. mexicana for 16h. c-MET expression was assessed by flow cytometry. The representative MFI is shown. Data are representative of ≥ 2 experiments, n≥3.(TIF)Click here for additional data file.S4 FigA) Representative histology pictures of naïve liver stained with c-MET (green), F4/80 (white) and DAPI (blue) on the left. Liver stained in absence of the first step c-MET and F4/80 antibodies on the right, as control for primary antibodies specificity. Scale bar: 30μm. B) Representative pictures of Mrp8;Metwt/wt and Mrp8;Metfl/fl ears 6 hours p.i with 106L. mexicana, staining for phospho-c-MET , Ly6G (red) and DAPI (blue). Scale bars: 30μm. Note the absence of P-c-MET staining in neutrophils in Mrp8;Metfl/fl ears. Scale bar: 10μm. C) Representative histology picture of six weeks infected ear, showing immunofluorescent staining for MR+ dermal macrophages (red), c-MET (green) and DAPI+ nuclei (blue). Scale bar: 30μm (left), enlargement of the defined area (right), scale bars: 10μm. Data are representative of ≥1 experiments.(TIF)Click here for additional data file.S5 FigA) C57BL/6 mice were infected i.d. with 106L. mexicana metacyclic promastigotes. Twenty-four hours late, the infected ears were collected and homogenized in RIPA buffer using a tissue lyser and the level of HGF analysed by ELISA. PBS-injected ears were used as a control. B) Mrp8;Metfl/fl mice deficient for c-MET in PMNs and Mrp8;Metwt/wt control littermate were infected with 106L. mexicana metacyclic promastigotes in the ear dermis. Twenty-four hours post infection, ears were collected and digested to obtain a cell suspension. The frequency of CD45+CD11b+Ly6G+ PMNs was assessed by flow cytometry. Data are representative of ≥2 independent experiments, n≥5/group.(TIF)Click here for additional data file.


Antibiotic treatment for dengue is likely considerable and potentially avoidable but has not been well characterized. This study aimed to assess antibiotic prescribing for confirmed dengue cases in outpatient and inpatient settings and to identify associated patient, physician and contextual factors.57,301 adult dengue cases reported in Taiwan between 2008–2015 were analyzed. We assessed both outpatient and inpatient claims data of dengue patients from a week before to a week after their dengue infections were confirmed under Taiwan’s National Health Insurance program. A multivariable logistic regression with generalized estimating equations was used to estimate the probability of antibiotic prescribing in dengue patients.Overall, 24.6% of dengue patients were prescribed an antibiotic during the 14 day-assessment period. Antibiotics were prescribed in 6.1% and 30.1% of outpatient visits and inpatient admissions, respectively. Antibiotic prescriptions were reduced by ~50% in epidemic years. Among inpatients, advanced age, females, and major comorbidities were risk factors for receipt of an antibiotic; antibiotics were used in 26.0% of inpatients after dengue was diagnosed. Significant differences in antibiotic prescribing practices were observed among physicians in outpatient settings but not in inpatient settings.In addition to patient and physician demographic characteristics, contextual factors such as care setting and during epidemics significantly influenced prescription of antibiotics. Characterization of prescribing patterns should help direct programs to curb antibiotic prescribing. Antimicrobial resistance is a growing global public health threat. The non-specific clinical manifestations of dengue overlap with signs and symptoms of other febrile illnesses common to tropical and subtropical zones making differential diagnosis between dengue and bacterial infections difficult, hence, leading to potentially unnecessary antibiotic prescribing. However, our understanding of factors underlying antibiotic prescribing for dengue is rather limited. Taiwan has experienced periodic dengue outbreaks and has a comprehensive national health insurance database including reliable infectious diseases surveillance and prescribing records. The findings in Taiwan show that other than commonly known patient characteristics, provider and contextual factors play a significant role. Physician’s age and practice setting were significant factors influencing the decision to prescribe antibiotics, particularly in outpatient visits. The likelihood of prescribing an antibiotic to dengue patients was reduced by more than 50% in medical visits occurring after the dengue infection was confirmed. Understanding patient, provider and contextual factors in antibiotics prescription for dengue infections can provide insights for improved antibiotic stewardship and unnecessary antibiotic treatment for dengue. Growing antimicrobial resistance is recognized as an urgent threat to global health. Among other drivers, such as their promiscuous use in animal husbandry, widespread misuse and overuse of antibiotics in healthcare is a major contributor to the emergence of resistant pathogens . While aPoint-of-care dengue diagnostic assays are not available universally and their performance also depends on the local epidemiology of dengue and other flaviviral infections that can produce cross-reactions. As over half of the global population in at least 120 countries, some four billion people, is at risk for dengue, that antibiotic consumption is likely to be considerable .Compared to a large body of literature describing inappropriate antibiotic prescribing for acute viral respiratory illnesses such as colds, upper respiratory tract infections and bronchitis –21, therThis study was reviewed and approved by the Institutional Review Board of National Yang Ming Chiao Tung University (NO. YM109083E).Since the study was done using administrative datasets without identifiable personal information, individual informed consent was waived. Linked data were obtained from the Notifiable Disease Dataset of Confirmed Cases (NDDCC), operated by the Taiwan Centers for Disease Control (CDC), and the National Health Insurance Research Database (NHIRD) in Taiwan. The Communicable Disease Control Act requires clinicians to report every individual who is infected by or suspected of a notifiable communicable disease, including dengue, to the NDDCC, providing the most accurate source of confirmed dengue cases. The NDDCC was then linked to the NHIRD to obtain medical care utilization information on confirmed cases, including diagnoses, type of medication prescribed, patient and physician characteristics. All these databases are available for research purposes and managed by the Health and Welfare Data Center (HWDC) and linked using encrypted identification numbers of individual patients, physicians, and hospitals. All the analyses were conducted onsite at the HWDC. The validity and quality of the NHIRD have been assessed repeatedly and found to be satisfactory and well-suited for research purposes ,27.This was a population-based pooled cross-sectional study. The study population included all confirmed dengue cases in persons 18 years and older during the study period, 2008–2015 . The dengue cases in our sample were defined as dengue cases confirmed by the Taiwan CDC and recorded in the NDDCC dataset. In Taiwan, any suspected dengue case is required to be reported to the Taiwan CDC and his/her specimen also must be sent to Taiwan CDC for confirmation through Taiwan’s official infectious disease surveillance system. The cases confirmed by Taiwan CDC are recorded in the NDDCC dataset. Multiple dengue records of the same individual within 3 months were considered the same dengue case and only the initial report was analyzed. Each dengue case was assessed for his/her utilization of outpatient care, inpatient services, and antibiotics prescribed during a 14-day assessment period (from a week before to a week after the index date). The index date was defined as the individual’s date of confirmed dengue diagnosis recorded in Taiwan CDC’s database. Due to heterogeneity in disease progression, frequency of comorbidities, and factors influencing medical care utilization patterns between adults and children/adolescents, we did not include pediatric populations in our study. People with incomplete information on important characteristics or without any medical visit during the assessment period were excluded . The final study population included 57,301 dengue cases.Our main dependent variable was the presence or absence of at least one antibiotic prescription for each confirmed dengue case during the 14-day assessment period. Medical utilization patterns included antibiotics prescribed, outpatient visits, and hospitalization events. The medical utilization patterns included any antibiotic prescribed, any outpatient visit incurred, and any hospitalization incurred. All three types of medical service utilization were assessed during the 14-day assessment period. Any antibiotics prescribed, outpatient visit, or hospitalization occurring during the 14-day period, 7 days before confirmation, 7 days after confirmation were assessed. The frequency of specific antibiotics prescribed was calculated. Antibiotics prescribed in medical visits involving a concomitant bacterial infection diagnosis for which antibiotic treatment may be indicated were excluded for analyses . ConcomiPatient characteristics included patient’s age, sex, socioeconomic status (SES), comorbidity status, and year of confirmed dengue diagnosis. Patient’s age was classified into three groups: 18–45, 45–65, and above 65 years. SES was defined by the individual’s insurable wages and types. Patients were classified into those with and without regular well-defined monthly wage and the former were then categorized into two groups: monthly wage above or below NTD $30,000 . Comorbidity status was defined by presence of major comorbidities such as cancer, chronic kidney diseases, and others . We alsoPhysician characteristics included age, sex, specialty, and patient volume. Physician’s age was classified into three groups: under 40, 40–60, and above 60 years. Specialty was classified into five categories: family medicine, internal medicine, emergency, otolaryngology (ENT), and others. Patient volume was calculated separately for outpatient visits and inpatient admissions. Patient volume was defined as the average monthly number of ambulatory care visits or inpatient admissions one year before the index date. Both ambulatory and inpatient volumes were then categorized into tertiles as low, medium, and high. Characteristics of physician’s practice setting included ownership (public vs. private), accreditation level and urbanization of practice . We classified medical facilities into three levels: medical centers, regional and district hospitals, and clinics according to Taiwan’s medical facility accreditation regulation and process. Medical centers are the tertiary care hospitals which has met the highest quality of care standard under the current accreditation regulation.Descriptive statistics were computed to describe the characteristics of dengue patients. Prescribing patterns in outpatient and inpatient settings were analyzed separately. Since a dengue diagnostic test result could have influenced the antibiotic prescribing decision, we analyzed antibiotic prescribing in dengue patients before and after their dengue infection was confirmed. Due to the hierarchical nature of the data, cluster effects are likely; generalized estimating equation (GEE) was applied to control the probable clustering effects of visits within each physician. Robust estimators were applied for standard errors in all analyses. Sensitivity analyses were conducted: (1) instead of analyzing medical visits without a concomitant bacterial diagnosis, we tried different inclusion criteria ; (2) a shorter assessment period (6-day period from 3 days before to 3 days after the index date). The results of these sensitivity analyses remained robust. All analyses were conducted using SAS 9.4.Of the 57,301 confirmed adult dengue cases during the study period (2008–2015), 89.4% of cases occurred during the 2014–2015 epidemic . 21.5% oAntibiotics were prescribed in 6.1% of outpatient visits during the 14-day assessment period . The proAfter adjusting for other characteristics and clustering effects, prescribing patterns were relatively consistent for total visits, pre-confirmation visits and post-confirmation visits . SignifiAfter adjusting for patient characteristics, antibiotic prescribing in the outpatient setting varied significantly among physicians. Antibiotic prescribing increased with physician age. Compared to family medical physicians, Otolaryngology , emergency  and other specialists  were significantly more likely to order antibiotics. Among practice settings, physicians in public hospitals were 20% less likely to prescribe antibiotics (95% CI: 0.71–0.91) than in private settings; at a facility level, antibiotics were most likely to be prescribed in clinics  compared to all levels of hospitals.The most commonly prescribed antibiotics for dengue outpatients were mainly cephalosporins  and tetracyclines  compared to 21.8% by older physicians (60 years or above). Patients who were hospitalized after dengue was confirmed (26.0%) received antibiotic prescriptions less frequently than those hospitalized before confirmation (52.3%). Specific antibiotics ordered for patients hospitalized before and after the confirmation of dengue were similar.After adjusting for other characteristics and clustering effects, prescribing patterns were relatively consistent for all inpatient admissions . LikelihThis population-based study of antibiotic prescribing patterns for dengue in Taiwan between 2008–2015 found several associations of interest. Significantly, more than 1 in 4 dengue patients received at least one antibiotic prescription during the 14-day assessment period (i.e. from 7 days before to 7 days after dengue was confirmed). The proportion was significantly lower for both inpatients and outpatients in the two epidemic years than in nonepidemic years, indicating the underestimation of dengue in physicians’ pre-test assessments. Antibiotics were prescribed more frequently in inpatient (30.1%) than in ambulatory care settings (6.1%) with usage higher for older patients, females and patients with underlying conditions. In examining physician characteristics associated with antibiotic prescription, older physician age and private practice were significant factors influencing the decision to prescribe antibiotics in outpatient visits. The likelihood of prescribing an antibiotic to dengue patients was reduced by more than 50% in medical visits occurring after the dengue infection was confirmed.Antibiotic prescribing for dengue in outpatient visits (6.1%) was not as widespread as that observed for common viral respiratory illnesses such as acute bronchitis in Taiwan ,30,31, aConsistent with previous findings ,32, our Diagnostic uncertainty is a principal driver of physician decision-making in prescribing antibiotics . For denConsistent with previous studies, patient’s age, gender and comorbidity status play significant roles in prescribing decisions in both outpatient and inpatient care settings. As seen previously ,39, femaSignificant prescribing variations were observed among ambulatory care physicians across age and specialty, which may reflect differences in medical education and training of the respective cohorts. The trend for greater frequency of antibiotic prescribing with older age may be intuitive as attention to antimicrobial resistance in medical school education and residency training has been greater in recent decades. In addition to physician’s age and specialty, accreditation level and ownership of physician’s practice setting independently contributed to the decision. Physicians practicing in more technologically advanced hospitals were significantly more cautious in prescribing antibiotics than their counterparts in local clinics, possibly due to easier access to laboratory facilities, stronger demand for compliance to up-to-date guidelines, stricter continuing education requirements and peer pressure ,30,40. PAmong the study limitations, as NHI claims data capture antibiotics prescribed under the NHI program, out-of-pocket purchases by patients could not be included, leading to an underestimation of antibiotic use. Secondly, although we excluded visits with a concomitant bacterial infection, as outpatient claims only allow up to 3 diagnoses, bacterial infection or other conditions for which antibiotics may be indicated may not have been captured fully, leading to an overestimation. Third, as we did not review individual medical records and due to data limitation of the NHI claims data, we lacked more detailed clinical information to ascertain appropriateness of and indications for antibiotic prescribing. Therefore, to be conservative, we excluded all visits with bacterial infection from our main analysis and solely focused on describing potentially unnecessary antibiotic prescribing among dengue cases. Future research with more detailed clinical data may help in this regard. Fourth, confounding bias may have been introduced as data on laboratory results, clinical presentation and symptoms, disease severity, and physician-patient communication were lacking, impeding detailed investigations into rationales behind physicians’ prescribing decisions. Finally, because the study was conducted in a setting under a national health insurance program with easy access to providers and prescription drugs, the results may not apply to countries with different levels of resources or different health care systems.In conclusion, this population-based study of antibiotic prescribing for dengue patients systematically investigated a wide array of patient, physician, contextual factors and identified potentially modifiable circumstances that could be targeted for improved antibiotic stewardship. In Taiwan, at least, a “one-size fits all” antibiotic stewardship approach may not be effective in reducing unnecessary antibiotic prescribing for dengue. The aggregate result also provides a baseline for antibiotic use that hypothetically could be avoided when improvements in vector control and, potentially, introduction of effective vaccines reduce or even eliminate dengue transmission.S1 TextTable A in S1 Text. The average length of hospital stay among patients who had been hospitalized . Table B in (DOCX)Click here for additional data file.


Immunoglobulin (Ig)G4 is a unique protein molecule and its role in autoimmune diseases remains elusive and controversial. Accumulating evidence suggests a pathogenic role of IgG4 in rheumatoid arthritis (RA). Rheumatoid factors (RF) in RA can recognize the Fc domains of IgG4 to form RF-IgG4 immune complexes that may activate the complement system leading to synovial injury. The aim of this article was to systematically review the literature from the past 2 decades to determine the frequency of elevated IgG4 and its clinical significance in RA. We comprehensively searched the Pubmed, Scopus, and Web of Science databases with the following terms: “IgG4”, “rheumatoid arthritis”, and “immunoglobulin G4”, and scrutinized all of the relevant publications. Based on the selection criteria, 12 studies were incorporated, which involved a total of 1715 RA patients. Out of 328 subjects from three studies, the pooled frequency of elevated non-specific IgG4 was 35.98%. There was a significant positive correlation between the IgG4 levels and the RA disease activity based on DAS-28 measurements (r = 0.245–0.253) and inflammatory markers, i.e., erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels (r = 0.262–0.389). Longitudinal studies that measured the serial levels of IgG4 consistently showed a decline in the concentrations (up to 48% less than baseline) with disease modifying anti-rheumatic drug (DMARD) treatment. Current evidence suggests that serum IgG4 levels are significantly elevated in RA compared to the general population. This review indicates that IgG4 is a promising biomarker of disease activity and tends to decline in response to DMARD therapies. Biologic therapies have revolutionized the therapeutic armamentarium of RA in the recent decade, and IgG4 appears to be a potential treatment target. Immunoglobulin (Ig)G accounts for 80% of the total immunoglobulins in human serum, and can be divided into four subclasses, i.e., IgG1 60–70%), IgG2 15–20%), IgG3 (5–10%), and IgG4 (4–6%). Each of these has different immunological properties and functions [–20%, IgG0–70%, IgIgG4 is a poorly understood molecule with controversial roles in the immune system. Traditionally, IgG4 has been viewed as a “non-inflammatory” molecule, which dampens rather than incites immune activation. This is due to the unique molecular structure of IgG4, whereby the heavy chains in each IgG4 molecule have inefficient disulphide bridges due to a single amino acid difference in the hinge region . In hemiNevertheless, accumulating evidence suggests a pathogenic role of IgG4 based on its correlations with disease activity and the severity of certain disease entities . IgG4 maAs far as we know, there are no published systematic reviews focusing on IgG4 in RA. Hence, the purpose of this systematic review was to gather and scrutinize all available literature in the past few decades to determine the pooled frequency of elevated IgG4 and its clinical significance. We comprehensively searched the Pubmed, Scopus, and Web of Science databases with the following terms: “IgG4”, “immunoglobulin G4”, and “rheumatoid arthritis”, and tracked all of the publications. All three authors independently performed a literature search by title and abstract screening using the Endnote software. In the event of uncertainty, the full text of the article was obtained and assessed. Disagreements were resolved by a consensus-based discussion. Only articles that were approved after much scrutiny by all were finally included in the review. To minimize the selection, information, and confounding biases, the PICOT  approach was employed to develop the inclusion and exclusion criteria . The popAll adult human studies written in English that looked into IgG4 in RA were included. Conference abstracts with sufficient data were considered eligible. We excluded studies published before 2000. Furthermore, articles in other languages, case reports, case series, animal studies, editorials, and review articles were excluded.After compiling the relevant studies, the authors extracted the relevant data from each paper, including year of publication, country, study design, study population, frequency of subjects with elevated IgG4, mean/median IgG4 levels in RA, and the correlations with clinical and biochemical markers. The Newcastle−Ottawa Scale  Table 1Table 1 wBased on the selection criteria, 12 studies were incorporated, which involved a total of 1715 RA patients. Among the included twelve research works, four were from Asia ,15,19,20IgG4 levels were detected using three methods, i.e., ELISA in seven studies ,26,27,28There were five studies that analyzed the non-specific IgG4 levels in RA ,19,20,23There were four studies that investigated the levels of IgG4 specific to citrullinated cyclic peptide (CCP) ,22,27,28p = 0.016), and with ESR . In keeping with these findings, Chen et al. [p < 0.05) [There were four studies that investigated the association of serum IgG4 levels with the RA disease activity ,15,19,20 < 0.05) . There w2 and a median ratio of IgG4+/IgG+ plasma cells of 19.1 (8.4–31.5). Both of them were positively correlated with ESR, CRP, and serum IgG4  [In one of the studies, the synovial samples of RA patients had a median IgG4 positive(+) plasma cells count of 83 (10–192)/mm < 0.05) . There were four longitudinal studies ,23,26,28Among the subjects who were treated with adalimumab, secondary failure to this biologic therapy was due to the formation of anti-drug antibody, which was IgG4 in up to 29% of the subjects .To the best of our knowledge, this is the first systematic review in the literature on IgG4 in RA. Our pooled analyses showed that IgG4 levels were significantly elevated in RA patients (35.98%) compared to the frequencies reported in healthy individuals. Various studies have found that the frequency of elevated IgG4 in healthy subjects ranged from 0–2.5% ,31. In kThere was a significant positive correlation between the IgG4 levels and RA disease activity based on the findings of all three studies that performed correlation analyses between the above-mentioned parameters. Of note, all three studies used the same composite clinical disease activity tool, i.e., DAS28-ESR, which may partially explain the similarity in the findings. Disease activity in RA reflects synovial inflammation, which is due to the effects of circulating cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) α. The synthesis of IgG4 in vitro was regulated by IL-6. IL-6 may enhance IgG4 production through IL-21 expressed in CD4+ T cells , which iThe independent role of IgG4 in RA remains elusive, although there is some supporting evidence based on the histopathological analysis by Chen et al. ,15. The Rheumatoid factors (RF) in RA can recognize the Fc domains of IgG4 to form RF-IgG4 immune complexes that may activate the complement system, leading to synovial injury . AlthougThe evidence from this systematic review suggests that IgG4 is a reliable biomarker of treatment response. The results from the studies in this regard were consistent. There are a few hypothetical explanations for the above. DMARD therapy tends to inhibit IgG4 production via TNFα inhibition . FurtherWe acknowledge the limitations of this systematic review. Most of the studies were conducted in Europe and Asia, which may limit the representativeness of the results to a certain extent. There are racial and ethnic disparities with regard to the disease characteristics and clinical outcomes in RA . We wereCurrent evidence suggests that the serum IgG4 levels are elevated in RA compared to the general population. This review indicates that IgG4 is a promising biomarker of disease activity, and tends to decline in response to DMARD therapies. Thus, IgG4 could serve as an alternative modality in RA to assess patients’ disease severity. There are several theories with regard to the pro-inflammatory role of IgG4. Further research is necessary to substantiate these hypotheses.