RESUMO
Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect â¼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.
Assuntos
Antibacterianos/farmacologia , Wolbachia/efeitos dos fármacos , Animais , Filariose Linfática/tratamento farmacológico , Filariose Linfática/microbiologia , Feminino , Masculino , Camundongos , Camundongos SCID , Oncocercose/tratamento farmacológico , Oncocercose/microbiologia , Pirimidinas/farmacologia , Quinazolinas/farmacologiaRESUMO
A type 1 immune response is involved in atherosclerosis progression, whereas the role of a type 2 polarization, especially with regard to an enhanced T helper (Th)2 cell differentiation, is still unclear. Helminths trigger type 2 immune responses, protecting the host from inflammatory disorders. We investigated whether an increased type 2 polarization by administration of Litomosoides sigmodontis adult worm extract (LsAg) affects atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Injections of 50 µg LsAg, i.p. into ApoE-/- mice induced a type 2 immune response shown by increased frequencies of peritoneal eosinophils and alternatively activated macrophages. To analyze the effect of LsAg on atherosclerosis initiation, ApoE-/- mice received a high-fat diet for 12 wk and weekly injections of 50 µg LsAg from wk 5 to 12. Therapeutic effects on advanced atherosclerosis were analyzed in mice that were fed a high-fat diet for 12 wk followed by 12 wk of normal chow and weekly LsAg injections. Both preventive and therapeutic LsAg application significantly decreased plaque size. Therapeutic treatment even caused regression of plaque size and macrophage density in the aortic root and reduced Th1-specific gene expression and intraplaque inflammation. In addition, plaque size after therapeutic treatment was inversely correlated with plaque-infiltrated alternatively activated macrophages. In vitro, LsAg treatment of HUVECs reduced intracellular levels of phosphorylated NF-κB-p65, IκB-α, and JNK1/2. In bifurcation flow-through slides, THP-1 cell adhesion to a HUVEC monolayer was decreased by LsAg in regions of nonuniform shear stress. Applying inhibitors of the respective kinases suggests JNK1/2 inhibition is involved in the suppressed cell adhesion. A switch to an enhanced type 2 immune response by LsAg exerts antiatherogenic effects on murine plaque development, indicating a protective role of a hampered type 1 polarization. In vitro, LsAg affects endothelial signaling pathways, among which JNK1/2 inhibition seems to be involved in the suppression of monocytic cell adhesion under proatherogenic shear stress.-Constanze, K., Tauchi, M., Furtmair, R., Urschel, K., Raaz-Schrauder, D., Neumann, A.-L., Frohberger, S. J., Hoerauf, A., Regus, S., Lang, W., Sagban, T. A., Stumpfe, F. M., Achenbach, S., Hübner, M. P., Dietel, B. Filarial extract of Litomosoides sigmodontis induces a type 2 immune response and attenuates plaque development in hyperlipidemic ApoE-knockout mice.
Assuntos
Aterosclerose/tratamento farmacológico , Misturas Complexas , Filarioidea/química , Hiperlipidemias/tratamento farmacológico , Placa Aterosclerótica/tratamento farmacológico , Células Th2/imunologia , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/imunologia , Misturas Complexas/química , Misturas Complexas/farmacologia , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/genética , Hiperlipidemias/imunologia , Camundongos , Camundongos Knockout para ApoE , Placa Aterosclerótica/induzido quimicamente , Placa Aterosclerótica/imunologia , Células Th1/imunologia , Células Th1/patologiaRESUMO
Microglia mediated responses to neuronal damage in the form of neuroinflammation is a common thread propagating neuropathology. In this study, we investigated the microglial alterations occurring as a result of sphingosine 1-phosphate (S1P) accumulation in neural cells. We evidenced increased microglial activation in the brains of neural S1P-lyase (SGPL1) ablated mice (SGPL1fl/fl/Nes ) as shown by an activated and deramified morphology and increased activation markers on microglia. In addition, an increase of pro-inflammatory cytokines in sorted and primary cultured microglia generated from SGPL1 deficient mice was noticed. Further, we assessed autophagy, one of the major mechanisms in the brain that keeps inflammation in check. Indeed, microglial inflammation was accompanied by defective microglial autophagy in SGPL1 ablated mice. Rescuing autophagy by treatment with rapamycin was sufficient to decrease interleukin 6 (IL-6) but not tumor necrosis factor (TNF) secretion in cultured microglia. Rapamycin mediated decrease of IL-6 secretion suggests a particular mechanistic target of rapamycin (mTOR)-IL-6 link and appeared to be microglia specific. Using pharmacological inhibitors of the major receptors of S1P expressed in the microglia, we identified S1P receptor 2 (S1PR2) as the mediator of both impaired autophagy and proinflammatory effects. In line with these results, the addition of exogenous S1P to BV2 microglial cells showed similar effects as those observed in the genetic knock out of SGPL1 in the neural cells. In summary, we show a novel role of the S1P-S1PR2 axis in the microglia of mice with neural-targeted SGPL1 ablation and in BV2 microglial cell line exogenously treated with S1P.
Assuntos
Aldeído Liases/metabolismo , Autofagia/fisiologia , Inflamação/metabolismo , Microglia/metabolismo , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/genética , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Inflamação/patologia , Interleucina-6/metabolismo , Camundongos Transgênicos , Microglia/patologia , Receptores de Esfingosina-1-Fosfato/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-ß RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.
RESUMO
Group 2 innate lymphoid cells (ILC2s) are inducers of type 2 immune responses, but their role during filarial infection remains unclear. In the present study, we used the Litomosoides sigmodontis rodent model of filariasis to analyze ILC2s during infection in susceptible BALB/c mice that develop a chronic infection with microfilaremia and semi-susceptible C57BL/6 mice that eliminate the filariae shortly after the molt into adult worms and thus do not develop microfilaremia. ILC2s (CD45+ Lineage- TCRß- CD90.2+ Sca-1+ IL-33R+ GATA-3+) were analyzed in the pleural cavity, the site of L. sigmodontis infection, after the infective L3 larvae reached the pleural cavity (9 days post infection, dpi), after the molt into adult worms (30dpi) and during the peak of microfilaremia (70dpi). C57BL/6 mice had significantly increased ILC2 numbers compared to BALB/c mice at 30dpi, accompanied by substantially higher IL-5 and IL-13 levels, indicating a stronger type 2 immune response in C57BL/6 mice upon L. sigmodontis infection. At this time point the ILC2 numbers positively correlated with the worm burden in both mouse strains. ILC2s and GATA-3+ CD4+ T cells were the dominant source of IL-5 in L. sigmodontis-infected C57BL/6 mice with ILC2s showing a significantly higher IL-5 expression than CD4+ T cells. To investigate the importance of ILC2s during L. sigmodontis infection, ILC2s were depleted with anti-CD90.2 antibodies in T and B cell-deficient Rag2-/- C57BL/6 mice on 26-28dpi and the outcome of infection was compared to isotype controls. Rag2-/- mice were per se susceptible to L. sigmodontis infection with significantly higher worm burden than C57BL/6 mice and developed microfilaremia. Depletion of ILC2s did not result in an increased worm burden in Rag2-/- mice, but led to significantly higher microfilariae numbers compared to isotype controls. In conclusion, our data demonstrate that ILC2s are essentially involved in the control of microfilaremia in Rag2-/- C57BL/6 mice.
Assuntos
Filarioidea , Imunidade Inata , Animais , Proteínas de Ligação a DNA , Suscetibilidade a Doenças , Interleucina-5 , Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis.
Assuntos
Brugia Malayi , Depsipeptídeos , Filariose Linfática , Loíase , Animais , Gatos , CãesRESUMO
Even after successful weight reduction, obese adults tend to quickly regain the lost weight. This raises the question of whether weight loss improves the underlying chronic adipose tissue inflammation characteristic of obesity. In order to improve our understanding of the mechanisms that reshape metabolic organs during weight loss, we investigated the macrophage and T cell function of the liver and adipose tissue on reversing high fat diet (HFD) mice to normal control diet (NCD). Obese mice that were switched to NCD showed an improvement in their metabolic profile that included enhanced glucose and insulin tolerance, decreased cholesterol, triglyceride, serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) levels that were comparable to NCD controls. However, despite weight loss, increased frequencies, but not total numbers, of IL-17+ and IL-22+ CD4+ T cells, IFN-γ+ and TNF+ CD8+ T cells and IL-17+ and IL-22+ CD8+ T cells were observed in the adipose tissue of mice switched from HFD to NCD compared to NCD and even HFD fed mice. Further, in the liver, IFN-γ+ and TNF+ CD8+ T cell, IL-17+ and IL-22+ CD8+ T cell, macrophage frequencies and their expression of antigen presenting molecules were increased. To determine if macrophages are the major determinants of the sustained inflammation observed during weight loss, we depleted macrophages, which significantly reduced IFN-γ+, TNF+, IL-17+, and IL-22+ CD8+ T cell frequencies in the liver and the adipose tissue. In conclusion, we show that although weight loss improves the metabolic profile, there is an active and ongoing CD8+ T cell inflammation in liver and adipose tissue mediated by macrophages.
Assuntos
Tecido Adiposo/imunologia , Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Fígado/imunologia , Macrófagos/fisiologia , Obesidade/imunologia , Redução de Peso , Animais , Dieta Hiperlipídica/efeitos adversos , Inflamação/patologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologiaRESUMO
Neutrophils are essentially involved in protective immune responses against invading infective larvae of filarial nematodes. The present study investigated the impact of S100A8/S100A9 on protective immune responses against the rodent filarial nematode Litomosoides sigmodontis. S100A9 forms with S100A8 the heterodimer calprotectin, which is expressed by circulating neutrophils and monocytes and mitigates or amplifies tissue damage as well as inflammation depending on the immune environment. Mice deficient for S100A8/A9 had a significantly reduced worm burden in comparison to wildtype (WT) animals 12 days after infection (dpi) with infective L3 larvae, either by the vector or subcutaneous inoculation, the latter suggesting that circumventing natural immune responses within the epidermis and dermis do not alter the phenotype. Nevertheless, upon intradermal injection of L3 larvae, increased total numbers of neutrophils, eosinophils and macrophages were observed within the skin of S100A8/A9-/- mice. Furthermore, upon infection the bronchoalveolar and thoracic cavity lavage of S100A8/A9-/- mice showed increased concentrations of CXCL-1, CXCL-2, CXCL-5, as well as elastase in comparison to the WT controls. Neutrophils from S100A8/A9-/- mice exhibited an increased in vitro activation and reduced L3 larval motility more effectively in vitro compared to WT neutrophils. The depletion of neutrophils from S100A8/A9-/- mice prior to L. sigmodontis infection until 5dpi abrogated the protective effect and led to an increased worm burden, indicating that neutrophils mediate enhanced protective immune responses against invading L3 larvae in S100A8/A9-/- mice. Interestingly, complete circumvention of protective immune responses in the skin and the lymphatics by intravenous injection of L3 larvae reversed the phenotype and resulted in an increased worm burden in S100A8/A9-/- mice. In summary, our results reveal that lack of S100A8/S100A9 triggers L3-induced inflammatory responses, increasing chemokine levels, granulocyte recruitment as well as neutrophil activation and therefore impairs larval migration and susceptibility for filarial infection.
Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Filariose/imunologia , Filarioidea/imunologia , Animais , Regulação da Expressão Gênica , Larva/imunologia , Pulmão/parasitologia , Pulmão/patologia , Camundongos , Camundongos Knockout , Neutrófilos/fisiologiaRESUMO
A major impediment to eliminate lymphatic filariasis and onchocerciasis is the lack of effective short-course macrofilaricidal drugs or regimens that are proven to be safe for both infections. In this study we tested oxfendazole, an anthelmintic shown to be well tolerated in phase 1 clinical trials. In vitro, oxfendazole exhibited modest to marginal motility inhibition of adult worms of Onchocerca gutturosa, pre-adult worms of Onchocerca volvulus and Onchocerca lienalis microfilariae. In vivo, five days of oral treatments provided sterile cure with up to 100% macrofilaricidal efficacy in the murine Litomosoides sigmodontis model of filariasis. In addition, 10 days of oral treatments with oxfendazole inhibited filarial embryogenesis in patent L. sigmodontis-infected jirds and subsequently led to a protracted but complete clearance of microfilaremia. The macrofilaricidal effect observed in vivo was selective, as treatment with oxfendazole of microfilariae-injected naïve mice was ineffective. Based on pharmacokinetic analysis, the driver of efficacy is the maintenance of a minimal efficacious concentration of approximately 100 ng/ml (based on subcutaneous treatment at 25 mg/kg in mice). From animal models, the human efficacious dose is predicted to range from 1.5 to 4.1 mg/kg. Such a dose has already been proven to be safe in phase 1 clinical trials. Oxfendazole therefore has potential to be efficacious for treatment of human filariasis without causing adverse reactions due to drug-induced microfilariae killing.
Assuntos
Benzimidazóis/farmacologia , Filariose Linfática/tratamento farmacológico , Filarioidea/efeitos dos fármacos , Animais , Anti-Helmínticos/uso terapêutico , Modelos Animais de Doenças , Filariose Linfática/parasitologia , Feminino , Filarioidea/embriologia , Gerbillinae/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microfilárias/efeitos dos fármacos , Onchocerca/efeitos dos fármacos , Onchocerca volvulus/efeitos dos fármacos , Oncocercose/tratamento farmacológicoRESUMO
Novel-antibiotics are urgently needed to combat an increase in morbidity and mortality due to resistant bacteria. The preclinical candidate corallopyronin A (CorA) is a potent antibiotic against Gram-positive and some Gram-negative pathogens for which a solid oral formulation was needed for further preclinical testing of the active pharmaceutical ingredient (API). The neat API CorA is poorly water-soluble and instable at room temperature, both crucial characteristics to be addressed and overcome for use as an oral antibiotic. Therefore, amorphous solid dispersion (ASD) was chosen as formulation principle. The formulations were prepared by spray-drying, comprising the water-soluble polymers povidone and copovidone. Stability (high-performance liquid chromatography, Fourier-transform-infrared spectroscopy, differential scanning calorimetry), dissolution (biphasic dissolution), and solubility (biphasic dissolution, Pion's T3 apparatus) properties were analyzed. Pharmacokinetic evaluations after intravenous and oral administration were conducted in BALB/c mice. The results demonstrated that the ASD formulation principle is a suitable stability- and solubility-enhancing oral formulation strategy for the API CorA to be used in preclinical and clinical trials and as a potential market product.
RESUMO
The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal-adult worm killing-drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10-14 days. Therefore, AN11251 represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis.
Assuntos
Antibacterianos/farmacologia , Diterpenos/farmacologia , Filariose/tratamento farmacológico , Filarioidea/efeitos dos fármacos , Compostos Policíclicos/farmacologia , Wolbachia/efeitos dos fármacos , Animais , Boro , Doxiciclina/farmacologia , Feminino , Filariose/microbiologia , Filarioidea/microbiologia , Camundongos Endogâmicos BALB C , Rifampina/farmacologia , Simbiose , PleuromutilinasRESUMO
The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16-18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10-14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days.
Assuntos
Antibacterianos/uso terapêutico , Filariose/tratamento farmacológico , Oncocercose/tratamento farmacológico , Quinazolinas/uso terapêutico , Wolbachia/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Brugia pahangi/efeitos dos fármacos , Feminino , Filariose/microbiologia , Filarioidea/efeitos dos fármacos , Gerbillinae/microbiologia , Gerbillinae/parasitologia , Microfilárias/efeitos dos fármacos , Quinazolinas/administração & dosagem , Simbiose/efeitos dos fármacosRESUMO
Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection.
Assuntos
Filariose/patologia , Filarioidea/imunologia , Inflamação/patologia , Pulmão/imunologia , Macrófagos/imunologia , Receptores de Interleucina-4/metabolismo , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/análise , Animais , Modelos Animais de Doenças , Feminino , Filariose/imunologia , Inflamação/imunologia , Pulmão/patologia , Macrófagos/química , Camundongos Endogâmicos BALB C , Células Th2/imunologiaRESUMO
Compared to the innate immune system, the contribution of the adaptive immune response during obesity and insulin resistance is still not completely understood. Here we demonstrate that high fat diet (HFD) increases the frequencies of activated CD4+ and CD8+ T cells and frequencies of T cells positive for IFN-γ and IL-17 in the adipose tissue. The adipocyte-derived soluble factor adiponectin reduces IFN-γ and IL-17 positive CD4+ T cells from HFD mice and dampens the differentiation of naïve T cells into Th1 cells and Th17 cells. Adiponectin reduces Th17 cell differentiation and restrains glycolysis in an AMPK dependent fashion. Treatment with adult worm extracts of the rodent filarial nematode Litomosoides sigmodontis (LsAg) reduces adipose tissue Th1 and Th17 cell frequencies during HFD and increases adiponectin levels. Stimulation of T cells in the presence of adipocyte-conditioned media (ACM) from LsAg-treated mice reduces Th1 and Th17 frequencies and this effect was abolished when ACM was treated with an adiponectin neutralizing antibody. Collectively, these data reveal a novel role of adiponectin in controlling pro-inflammatory CD4+ T cells during obesity and suggest that the beneficial role of helminth infections and helminth-derived products on obesity and insulin resistance may be in part mediated by adiponectin.
Assuntos
Adiponectina/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Interferon gama/biossíntese , Interleucina-17/biossíntese , Obesidade/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Dieta Hiperlipídica , Glucose/metabolismo , Glicólise , Insulina/metabolismo , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Obesidade/etiologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: Mice are susceptible to infections with the rodent filarial nematode Litomosoides sigmodontis and develop immune responses that resemble those of human filarial infections. Thus, the L. sigmodontis model is used to study filarial immunomodulation, protective immune responses against filariae and to screen drug candidates for human filarial diseases. While previous studies showed that type 2 immune responses are protective against L. sigmodontis, the present study directly compared the impact of eosinophils, IL-5, and the IL-4R on the outcome of L. sigmodontis infection. METHODS: Susceptible wildtype (WT) BALB/c mice, BALB/c mice lacking eosinophils (dblGATA mice), IL-5-/- mice, IL-4R-/- mice and IL-4R-/-/IL-5-/- mice were infected with L. sigmodontis. Analyses were performed during the peak of microfilaremia in WT animals (71 dpi) as well as after IL-4R-/-/IL-5-/- mice showed a decline in microfilaremia (119 dpi) and included adult worm counts, peripheral blood microfilariae levels, cytokine production from thoracic cavity lavage, the site of adult worm residence, and quantification of major immune cell types within the thoracic cavity and spleen. RESULTS: Our study reveals that thoracic cavity eosinophil numbers correlated negatively with the adult worm burden, whereas correlations of alternatively activated macrophage (AAM) numbers with the adult worm burden (positive correlation) were likely attributed to the accompanied changes in eosinophil numbers. IL-4R-/-/IL-5-/- mice exhibited an enhanced embryogenesis achieving the highest microfilaremia with all animals becoming microfilariae positive and had an increased adult worm burden combined with a prolonged adult worm survival. CONCLUSIONS: These data indicate that mice deficient for IL-4R-/-/IL-5-/- have the highest susceptibility for L. sigmodontis infection, which resulted in an earlier onset of microfilaremia, development of microfilaremia in all animals with highest microfilariae loads, and an extended adult worm survival.
Assuntos
Suscetibilidade a Doenças/imunologia , Eosinófilos/imunologia , Filariose/imunologia , Interleucina-5/genética , Receptores de Superfície Celular/genética , Animais , Modelos Animais de Doenças , Filariose/sangue , Filarioidea/fisiologia , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microfilárias/imunologia , Ácaros/parasitologia , Transdução de Sinais , Baço/imunologiaRESUMO
Depletion of Wolbachia endosymbionts of human pathogenic filariae using 4-6 weeks of doxycycline treatment can lead to permanent sterilization and adult filarial death. We investigated the anti-Wolbachia drug candidate ABBV-4083 in the Litomosoides sigmodontis rodent model to determine Wolbachia depletion kinetics with different regimens. Wolbachia reduction occurred in mice as early as 3 days after the initiation of ABBV-4083 treatment and continued throughout a 10-day treatment period. Importantly, Wolbachia levels continued to decline after a 5-day-treatment from 91.5% to 99.9% during a 3-week washout period. In jirds, two weeks of ABBV-4083 treatment (100mg/kg once-per-day) caused a >99.9% Wolbachia depletion in female adult worms, and the kinetics of Wolbachia depletion were recapitulated in peripheral blood microfilariae. Similar to Wolbachia depletion, inhibition of embryogenesis was time-dependent in ABBV-4083-treated jirds, leading to a complete lack of late embryonic stages (stretched microfilariae) and lack of peripheral microfilariae in 5/6 ABBV-4083-treated jirds by 14 weeks after treatment. Twice daily treatment in comparison to once daily treatment with ABBV-4083 did not significantly improve Wolbachia depletion. Moreover, up to 4 nonconsecutive daily treatments within a 14-dose regimen did not significantly erode Wolbachia depletion. Within the limitations of an animal model that does not fully recapitulate human filarial disease, our studies suggest that Wolbachia depletion should be assessed clinically no earlier than 3-4 weeks after the end of treatment, and that Wolbachia depletion in microfilariae may be a viable surrogate marker for the depletion within adult worms. Furthermore, strict daily adherence to the dosing regimen with anti-Wolbachia candidates may not be required, provided that the full regimen is subsequently completed.
Assuntos
Antibacterianos/farmacologia , Filarioidea/microbiologia , Microfilárias/microbiologia , Wolbachia/efeitos dos fármacos , Wolbachia/fisiologia , Animais , Doxiciclina/farmacologia , Feminino , Filariose , Filarioidea/efeitos dos fármacos , Gerbillinae , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Microfilárias/efeitos dos fármacos , Microfilárias/embriologia , Modelos AnimaisRESUMO
Flubendazole (FBZ) is highly efficacious against filarial nematodes after parenteral administration and presents a promising macrofilaricidal drug candidate for the elimination of onchocerciasis and other filariae. In the present study the efficacy of a newly developed bioavailable amorphous solid dispersion (ASD) oral formulation of FBZ was investigated in the Litomosoides sigmodontis jird model. FBZ was administered to chronically infected, microfilariae-positive jirds by single (40mg/kg), repeated (2, 6 or 15mg/kg for 5 or 10 days) oral (OR) doses or single subcutaneous (SC) injections (2 or 10mg/kg). Jirds treated with 5 SC injections at 10mg/kg served as positive controls, with untreated animals used as negative controls. After OR doses, FBZ is rapidly absorbed and cleared and the exposures increased dose proportionally. SC administered FBZ was slowly released from the injection site and plasma levels remained constant up to necropsy eight weeks after treatment end. Increasing single SC doses caused less than dose-proportional exposures. At necropsy, all animals receiving 1x or 5x 10mg/kg SC FBZ had cleared all adult worms and the 1x 2mg/kg SC treatment had reduced the adult worm burden by 98%. 10x 15mg/kg OR FBZ reduced the adult worm burden by 95%, whereas 1x 40mg/kg and 5x 15mg/kg OR reduced the worm burden by 85 and 84%, respectively. Microfilaremia was completely cleared at necropsy in all animals of the SC treatment regimens, while all oral FBZ treatment regimens reduced the microfilaremia by >90% in a dose and duration dependent manner. In accordance, embryograms from female worms revealed a FBZ dose and duration dependent inhibition of embryogenesis. Histological analysis of the remaining female adult worms showed that FBZ had damaged the body wall, intestine and most prominently the uterus and uterine content. Results of this study demonstrate that single and repeated SC injections and repeated oral administrations of FBZ have an excellent macrofilaricidal effect.
Assuntos
Filariose/tratamento farmacológico , Filaricidas/farmacologia , Filaricidas/farmacocinética , Filarioidea/efeitos dos fármacos , Mebendazol/análogos & derivados , Administração Oral , Animais , Modelos Animais de Doenças , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Filariose/parasitologia , Filaricidas/administração & dosagem , Filarioidea/embriologia , Gerbillinae/parasitologia , Mebendazol/administração & dosagem , Mebendazol/farmacocinética , Mebendazol/farmacologia , Carga ParasitáriaRESUMO
There is a significant need for improved treatments for onchocerciasis and lymphatic filariasis, diseases caused by filarial worm infection. In particular, an agent able to selectively kill adult worms (macrofilaricide) would be expected to substantially augment the benefits of mass drug administration (MDA) with current microfilaricides, and to provide a solution to treatment of onchocerciasis / loiasis co-infection, where MDA is restricted. We have identified a novel macrofilaricidal agent, Tylosin A (TylA), which acts by targeting the worm-symbiont Wolbachia bacterium. Chemical modification of TylA leads to improvements in anti-Wolbachia activity and oral pharmacokinetic properties; an optimized analog (ABBV-4083) has been selected for clinical evaluation.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Filaricidas/farmacologia , Tilosina/análogos & derivados , Tilosina/farmacologia , Wolbachia/efeitos dos fármacos , Animais , Antibacterianos/farmacocinética , Filariose Linfática/tratamento farmacológico , Feminino , Filaricidas/farmacocinética , Filarioidea/efeitos dos fármacos , Filarioidea/microbiologia , Gerbillinae , Camundongos , Camundongos Endogâmicos BALB C , Oncocercose/tratamento farmacológico , Simbiose/efeitos dos fármacosRESUMO
A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.
Assuntos
Boro/farmacologia , Diterpenos/farmacologia , Filariose Linfática/tratamento farmacológico , Filaricidas/uso terapêutico , Oncocercose/tratamento farmacológico , Compostos Policíclicos/farmacologia , Wolbachia/efeitos dos fármacos , Wuchereria bancrofti/efeitos dos fármacos , Animais , Boro/química , Diterpenos/química , Filaricidas/farmacocinética , Filaricidas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Compostos Policíclicos/química , PleuromutilinasRESUMO
Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets Wolbachia, a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in L. sigmodontis, Brugia malayi, and Onchocerca ochengi in vivo preclinical models of filarial disease and in vitro selectivity against Loa loa (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.