cAMP-induced arginase and their role in Mycobacterium tuberculosis growth: Emerging of new adjunctive therapies
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1
Louisiana State University Health Sciences Center-NO, Internal Medicine, United States
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2
Johns Hopkins University, Center for Tuberculosis Research, United States
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3
Louisiana State University Health Sciences Center-NO, Stanley S. Scott Cancer Center, United States
INTRODUCTION
One third of the world’s population is infected with Mycobaterium tuberculosis (Mtb). There are 9 million new cases of tuberculosis (TB) per year, resulting in approximately 2 million deaths. During the last decade, there has been a significant increase in the number of TB cases that may be the result of increased migration and/or the association of Mtb with HIV. On the other hand, barriers that negatively affect clinical outcomes such as the lengthy multidrug therapy dramatically affects compliance and adherence to medication, patient follow-up, pill burden and toxicity. This results in treatment failures and in the development of drug resistance. Regardless of its origin or negative barriers, the increases in TB burden promote an urgency to better understand host-pathogen and signaling pathways interactions that can lead to increase the susceptibility to TB to control the disease. We have gained new insights into why Mtb has won the battle against its control. Tuberculosis (TB) caused by infection with Mtb, remains as a major worldwide health treat. The standard treatment for TB is often effective, but it is also lengthy, complex, significantly toxic and can be thwarted by antibiotic resistance. Therefore, it is imperative to discover or develop new cost effective treatment strategies, especially in the current climate of drug-resistant tuberculosis. The successful replication, survival and persistence of Mtb in humans depend on its ability to survive inside host macrophages by subverting mechanisms of microbial killing. Upon Mtb infection, intracellular levels of 3’, 5’cyclic adenosine monophosphate (cAMP) increase in macrophages resulting in the induction of the enzyme arginase 2 (ARG2). Then, ARG2 metabolizes L-arginine to generate the production of L-ornithine, a precursor of polyamine synthesis which is necessary for cellular growth, Simultaneously, the increased production of ARG2 blocks the production of inducible nitric oxide synthase (NOS2) and nitric oxide (NO) a molecule in charge to control the growing of intracellular pathogens including Mtb. It has been demonstrated that ARG supports the growth of many parasitic and bacterial pathogens. Therefore, we believe that Mtb has developed the capacity to modulate the L-arginine metabolic pathway as a clever strategy to survive inside the macrophage.Thus, cAMP-induced overproduction of ARG2 may promote growth of Mtb at the expense of an impaired pathogen killing. Hence, the critical interplay between ARG and NOS2 might regulate the outcome of TB. The mechanisms of ARG2 induction in macrophages by Mtb are unknown, however, it appears that in mycobacterial infections, cAMP regulates signal transduction pathways responsible for ARG induction. We hypothesize that Mtb-derived cAMP triggers the induction of ARG and the synthesis of polyamines that are necessary for Mtb to survive. The blockage of cAMP and ARG signaling pathways with specific inhibitors could negatively affect Mtb growth and have a positive impact in the treatment of multi- and extensively-drug resistant tuberculosis.
OBJECTIVES
With this in mind, we plan to conduct experiments that will test the hypothesis that inhibition of polyamine synthesis suppress intracellular Mtb growth, and restore antimicrobial defenses by increasing NO production.
1. Determine if mycobacterial-cAMP regulates ARG induction within macrophages through a cAMP-PKA dependent mechanism, to promote Mtb growth.
2. Determine if inhibition of cAMP or ARG signaling, negatively affects Mtb growth and survival within the host.
METHODS
1. Human monocytes where obtained by negative selection after PBMC isolation. Three million monocytes were plated in six well plates in the presence of 5 ng/ml of hGM-CSF for 7 days to generate macrophages.
2. Macrophages were infected with Mtb-CDC1551 or Mtb-mutant (altered cAMP phenotype) at multiplicity of infection 1:3 for 24, 48 and 72 h were tissue culture supernatants and cell lysates were prepared for determination of arginase activity, arginase and NOS2 protein, cAMP, aminoacid levels (L-arginine, L-ornithine, L-glutamine, citrulline, polyamines), nitrites (NO), cytokines and bacterial culture. These elements were tested by enzymatic assays, western blot, ELISA, HPLC, Greiss reaction, multiplexing and colony forming units (CFU) respectively.
3. Adenylyl cyclases, protein kinase A (PKA), phosphodiesterases (PDE) 3,4,5, ARG, polyamine and NOS2 inhibitors were applied to the cultured macrophages 3 hours before and 3 hours after infection, to test for timing and effectiveness in Mtb control.
RESULTS
We found that there is a time dependent increase in cAMP levels in macrophages, that parallels with increases in ARG activity when human macrophages were infected with Mtb-CDC1551. PKA inhibitor H89 nearly abolished the induction of cAMP and therefore ARG activity. The inhibition of Mtb growth inside macrophages was markedly reduced in cells treated with H89 inhibitor. Adenylyl cyclase inhibitors were more effective when Mtb was treated before infection. There is a significant induction of ARG2 (p< 0.001) in macrophages infected with Mtb that parallels with increased Mtb growth 4.8, 5.16 and 6.4 log10 CFU at 24, 48 and 72 h respectively. Interestingly when we used the Mtb-mutant (lacking cAMP) we observed a significant decrease in cAMP (p=0.024), ARG (p=0.023) and Mtb growth, After 24 hours of infection there was a significant increase in NO production compared to uninfected cells. However, no increases in NO production were observed after 48 hours.
The addition of inhibitors nor-NOHA and BEC to the cultures, significantly inhibited Mtb growth (p= 0.005) associated with a significant decrease in the synthesis of polyamine (p=0.03) as compared to untreated cells. We also observed that ARG inhibition significantly increased the production of NO (p= 0.002).
There is a significant decrease in cAMP, ARG and Mtb growth when cells are treated with PDE5 inhibitor. The use of PED3 and IBMX (unspecific PDE inhibitor) presented opposite effect that those seen with PDE5 treatment.
CONCLUSIONS
1) This study shows in human monocytes-derived macrophages ARG2 is induced by mycobacterial cAMP. This increase in intracellular ARG induces the synthesis of polyamines necessary for Mtb growth and survival.
2) Induction of ARG blocks the production od NO as an other advantage for Mtb survival.
3) Blocking cAMP and ARG pathways, significantly decreases Mtb growth. The decrease in Mtb growth is associated with a decrease in cAMP production, ARG induction and polyamine synthesis.
4) These findings indicate that Mtb directly injects cAMP into the host cell, affecting host cell signaling events that culminate with the induction of ARG and the synthesis of polyamines providing an advantageous environment for Mtb growth and survival.
5) The investigation of cAMP secretion and ARG induction by Mtb and its effects on host cell signaling and antimicrobial responses
could reveal cellular events that are critical for Mtb survival providing a fresh perspective on how this pathogen establishes an infection and open new avenues for targeted treatments and/or prevention of tuberculosis.
6) Inhibitors of cAMP and ARG pathways can provide a new emerging therapy strategies to control Mtb growth specially in the
current environment of TB resistance. However, the results also clearly show that we need to be careful when using determined PDE inhibitors that can exacerbate the course of infection.
Acknowledgements
This project is supported by COBRE Grant P20 RR021970-05, NIH-NCRR and Stanley S. Scott Cancer Center Funds.
Keywords:
cAMP,
Macrophages,
Arginase2,
phospohodiesterase inhibition,
Mycobacterium tuberculosis,
Cytokines
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Oral Presentation
Topic:
Infectious and parasitic diseases
Citation:
Mason
C,
Porretta
E,
Bishai
W,
Ali
J and
Zea
AH
(2015). cAMP-induced arginase and their role in Mycobacterium tuberculosis growth: Emerging of new adjunctive therapies.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00302
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Received:
30 May 2015;
Published Online:
15 Sep 2015.
*
Correspondence:
Dr. Arnold H Zea, Louisiana State University Health Sciences Center-NO, Stanley S. Scott Cancer Center, New Orleans, Louisiana, 70112, United States, azea@lsuhsc.edu