RESUMO
Concerns regarding toxicity and resistance of current drugs in visceral leishmaniasis have been reported. Antimicrobial peptides are considered to be promising candidates and among them human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, in addition to its apoptosis-inducing role. Administration of hCAP18/LL-37 to infected macrophages also decreased parasite survival and increased the host favorable cytokine interleukin 12. However, 1,25-dihydroxyvitamin D3 (vitamin D3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the vitamin D3 receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection, resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. This study documents the antileishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Catelicidinas , Modulador de Elemento de Resposta do AMP Cíclico , Leishmania donovani , Leishmaniose Visceral , Macrófagos , Camundongos Endogâmicos BALB C , Leishmania donovani/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Humanos , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/tratamento farmacológico , Camundongos , Macrófagos/parasitologia , Macrófagos/metabolismo , Células THP-1 , Modulador de Elemento de Resposta do AMP Cíclico/metabolismo , Modulador de Elemento de Resposta do AMP Cíclico/genética , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Antiprotozoários/farmacologia , FemininoRESUMO
RNA fluorescence in situ hybridization (FISH) serves as a method for visualizing specific RNA molecules within cells. Its primary utility lies in the observation of messenger RNA (mRNA) molecules associated with particular genes of significance. This technique can also be applied to examine viral transcription and the localization of said transcripts within infected cells. In this context, we provide a comprehensive protocol for the detection, localization, and quantification of HIV-1 transcripts in mammalian cell lines. This encompasses the preparation of required reagents, cellular treatments, visualization, and the subsequent analysis of the data acquired. These parameters play a pivotal role in enhancing our comprehension of the molecular processes during infection, particularly at the crucial transcription phase of the viral life cycle.
Assuntos
HIV-1 , Hibridização in Situ Fluorescente , RNA Viral , Transcrição Gênica , Hibridização in Situ Fluorescente/métodos , Humanos , RNA Viral/genética , HIV-1/genética , RNA Mensageiro/genética , Infecções por HIV/virologia , Linhagem CelularRESUMO
The parasite Leishmania resides as amastigotes within the macrophage parasitophorous vacuoles inflicting the disease Leishmaniasis. Leishmania selectively modulates mitogen-activated protein kinase (MAPK) phosphorylation subverting CD40-triggered anti-leishmanial functions of macrophages. The mechanism of any pathogen-derived molecule induced host MAPK modulation remains poorly understood. Herein, we show that of the fifteen MAPKs, LmjMAPK4 expression is higher in virulent L. major. LmjMAPK4- detected in parasitophorous vacuoles and cytoplasm- binds MEK-1/2, but not MKK-3/6. Lentivirally-overexpressed LmjMAPK4 augments CD40-activated MEK-1/2-ERK-1/2-MKP-1, but inhibits MKK3/6-p38MAPK-MKP-3, phosphorylation. A rationally-identified LmjMAPK4 inhibitor reinstates CD40-activated host-protective anti-leishmanial functions in L. major-infected susceptible BALB/c mice. These results identify LmjMAPK4 as a MAPK modulator at the host-pathogen interface and establish a pathogen-intercepted host receptor signaling as a scientific rationale for identifying drug targets.