Neutrophils and dendritic cells (DCs) converge at localized sites of acute inflammation in the skin following pathogen deposition by the bites of arthropod vectors or by needle injection. Prior ...studies in mice have shown that neutrophils are the predominant recruited and infected cells during the earliest stage of Leishmania major infection in the skin, and that neutrophil depletion promotes host resistance to sand fly transmitted infection. How the massive influx of neutrophils aimed at wound repair and sterilization might modulate the function of DCs in the skin has not been previously addressed. The infected neutrophils recovered from the skin expressed elevated apoptotic markers compared to uninfected neutrophils, and were preferentially captured by dermal DCs when injected back into the mouse ear dermis. Following challenge with L. major directly, the majority of the infected DCs recovered from the skin at 24 hr stained positive for neutrophil markers, indicating that they acquired their parasites via uptake of infected neutrophils. When infected, dermal DCs were recovered from neutrophil depleted mice, their expression of activation markers was markedly enhanced, as was their capacity to present Leishmania antigens ex vivo. Neutrophil depletion also enhanced the priming of L. major specific CD4(+) T cells in vivo. The findings suggest that following their rapid uptake by neutrophils in the skin, L. major exploits the immunosuppressive effects associated with the apoptotic cell clearance function of DCs to inhibit the development of acquired resistance until the acute neutrophilic response is resolved.
Live whole-organism vaccines against Plasmodium falciparum malaria and cutaneous leishmaniasis remain the most uniformly effective vaccines against human parasitic diseases. These vaccines are ...discussed in terms of the requirement for persisting antigen to generate and maintain a protective response.
The origin and functional specialization of dermal macrophages in cutaneous infections have been little studied. In this paper, we show that a strain of
(
Seidman LmSd) that produces nonhealing ...cutaneous lesions in conventionally resistant C57BL/6 mice was more efficiently taken up by M2-polarized bone marrow (BM)-derived macrophages (BMDMs) in vitro and by mannose receptor (MR)
dermal macrophages in vivo compared with a healing strain (
Friedlin V1). Both in steady and in T helper type 1 (Th1) cell-driven inflammatory states, the MR
dermal macrophages showed M2 characteristics. The dermal macrophages were radio resistant and not replaced by monocytes or adult BM-derived cells during infection, but were locally maintained by IL-4 and IL-10. Notably, the favored infection of M2 BMDMs by LmSd in vitro was MR dependent, and genetic deletion of MR or selective depletion of MR
dermal macrophages by anti-CSF-1 receptor antibody reversed the nonhealing phenotype. We conclude that embryonic-derived, MR
dermal macrophages are permissive for parasite growth even in a strong Th1-immune environment, and the preferential infection of these cells plays a crucial role in the severity of cutaneous disease.
The phagosomal pathogen Leishmania appears unaffected by deliberate changes in the early Th1/Th2 balance. In this issue, Carneiro et al. explain these paradoxical results by showing that ...manipulations affecting IFN-γ-mediated phagocyte activation are counteracted by effects on IFN-γ-dependent recruitment of CCR2
monocytes permissive to parasite growth.
•Leishmania infection triggers activation of NLRP3 inflammasome in macrophages.•Inflammasome activation in macrophages restricts intracellular parasite replication.•Leishmania can partially inhibit ...inflammasome activation as an evasion strategy.•Inflammasome activation can promote pathology/parasite growth in certain conditions.
The inflammasomes are multi-molecular platforms that are activated in host cell cytoplasm when the innate immune cells are infected with pathogens or exposed to damage signals. Many independent groups reported that Leishmania infection trigger activation of the NLRP3 inflammasome in macrophages for restriction of intracellular parasite replication. Accordingly, Leishmania can dampen NLRP3 activation as an evasion strategy. In vivo, the NLRP3 inflammasome can promote parasite clearance, but the failure to eliminate parasites in the tissues together with sustained inflammasome activation can promote IL-1β-mediated disease pathology. In this review, we discuss the recent data regarding activation of the NLRP3 inflammasome in response to Leishmania and the beneficial and detrimental effects of the inflammasome during development of Leishmaniasis.
Tissue-resident macrophages are critical for tissue homeostasis and repair. We previously showed that dermis-resident macrophages produce CCL24 which mediates their interaction with IL-4
eosinophils, ...required to maintain their M2-like properties in the T
1 environment of the Leishmania major infected skin. Here, we show that thymic stromal lymphopoietin (TSLP) and IL-5
type 2 innate lymphoid cells are also required to maintain dermis-resident macrophages and promote infection. Single cell RNA sequencing reveals the dermis-resident macrophages as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permits specific labeling of dermis-resident macrophages and interstitial macrophages from other organs. Selective ablation of TSLP in dermis-resident macrophages reduces the numbers of IL-5
type 2 innate lymphoid cells, eosinophils and dermis-resident macrophages, and ameliorates infection. Our findings demonstrate that dermis-resident macrophages are self-maintained as a replicative niche for L. major by orchestrating localized type 2 circuitries with type 2 innate lymphoid cells and eosinophils.
Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a ...human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is "leishmanization," in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania.
Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), ...isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL‐1β mRNA and IL‐1β‐producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain, or caspase‐1/11, or lacking IL‐1β or IL‐1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen‐specific Th1 response. The possibility that IL‐1β might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL‐1β by LmSd‐infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase‐1. These data reveal that Nlrp3 inflammasome‐dependent IL‐1β, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.
Leishmania major infection drives NLPR3 inflammasome dependent IL‐1β secretion in infected macrophages, leading to early and persistent neutrophil recruitment to the lesion. Apoptotic neutrophils inhibit parasite clearance by suppressing the activation of macrophages for killing and dendritic cells for promoting Th1 development.
Sand flies are the natural vectors for the
species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/
interactions have been limited by the absence ...of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the
sand fly, a natural vector for
, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in
and were able to generate transmissible
mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with
Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for
parasites.
Sand flies are the natural vectors of
parasites. Studies of sand fly/
interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible
mutant alleles of
, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with
, showing that the sand fly immune response impacts its vector competence for this pathogen.
Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression, response to ...treatment and incidence of post-treatment sequelae, specifically post-kala-azar dermal leishmaniasis (PKDL). Nevertheless, the majority of infected people are asymptomatic carriers. Hamsters and mice are commonly used as models of fatal and non-fatal VL, respectively. Host and parasite genetics are likely to be important factors, but in general the reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Half of control hamsters had a rapid progression phenotype compared with none of the ABX-treated animals and the nine-month survival rate was significantly improved compared to untreated controls (40% vs. 10%). Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or haematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which Rodentibacter was specifically absent from ABX-treated hamsters, indicating this pathobiont may play a role in promoting disease progression. The results provide experimental support for antibiotic prophylaxis against secondary bacterial infections as an adjunct therapy in human VL patients.
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