Salmonella, melanoma and inflammasome activation: unraveling networks
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1
Instituto de Higiene, Universidad de la República, Departamento de Desarrollo Biotecnológico, Uruguay
Within the wide variety of cancers, melanoma stands out given the significant constant increase in its worldwide incidence and mortality. Even though melanoma is 20 times less common than other skin cancers (basal cell and squamous cell carcinomas), it is responsible for up to 90% of deaths from skin cancer (Avilés, 2006). Despite several years of extensive research, currently the only effective treatment is surgical resection of the primary tumor before it achieves thickness of 1mm and if not spread; whereas when the disease becomes metastatic treatment options are ineffective (Jack, 2006). However, recent substantial changes have been prompted in sight of better understanding of how the immune system might be modulated to fight melanoma in addition to the identification of several oncogenic mutations. Thus, new treatments for melanoma patients have emerged, particularly for the metastatic stage. In the last four years, Food and Drug Administration (FDA) has approved four new drugs to treat this stage of melanoma (reviewed in Shah, 2014). However, the modest increase in overall survival in addition to the low response rate (ipilimumab) or short duration of response (vemurafenib), high toxicity and the towering costs, highlight the urgent need of more effective and cheaper new therapies (Hodi, 2010; Burdine, 2014; Fellner, 2012; Menzies, 2013). In this regard, melanoma exhibits high immunogenicity, which makes it a good candidate for immunotherapies.
For many years, Salmonella has been evaluated for the treatment of cancer. Salmonella has intrinsic anti-tumor effect that together with the induction of a pro-inflammatory response could be a potent inductor of a specific anti-tumor immunity. Salmonella possesses many PAMPs, including flagellin, which has been demonstrated to be one of the bacteria major proinflammatory molecule (Zeng et al., 2003). Salmonella can activate inflammasome by two independent pathways. NLRC4 recognizes bacterial flagellin injected into the cytoplasm by type III secretion system (Franchi, 2006; Miao, 2006) meanwhile the precise signal that is detected by NLRP3 remains unclear. Moreover, it has been shown that the kinetics of assembly of both inflammasomes vary: NLRC4 is activated minutes after infection whereas NLRP3 activation happens many hours later. However, redundant roles for inflammasome receptors NLRP3 and NLRC4 have been described in host defense against Salmonella (Broz, 2010). Caspase-1 activation by most NLRs requires ASC, an adapter protein that is believed to bridge NLRs and caspase-1. It has been demonstrated that inflammasome activation plays an important role in innate immune defense against S. Typhimurium because mice lacking caspase-1, IL-1beta or IL-18 (the ultimate effectors of inflammasome activation pathway) succumb earlier to infection and have higher bacterial loads (Raupach, 2006).
Nevertheless, to our knowledge the interaction between Salmonella and inflammasome, and its role in Salmonella-mediated anti-tumor response have never been assessed. In the present work we intend to unravel this interaction, and particularly to study the role of flagellin, in vitro and in vivo in B16F1-melanoma model, and analyze its consequences on Salmonella well known anti-tumor effect. To achieve this aim, we constructed a Salmonella enterica serovar Typhimurium LVR01 aflagellated strain by silencing fliC and fljB genes, named LVR01flag-. We demonstrated that B16F1 cells do not express detectable levels of nlrp3 and nlrc4, even after stimulation with LVR01. However, a significant downregulation of asc and procaspase-1 inflammasome components was observed in B16F1 melanoma cell line in vitro, determined by a decrease in mRNA expression levels by qPCR. Interestingly this effect was completely abolished when the aflagellated strain LVR01flag- was used, both at early and late time points after infection (1h and 17h, respectively). In addition, il18, one of the final products of inflammasome cascade, also exhibits this behavior. Taken together these results could suggest that functional flagella plays a key role in the downregulation of inflammasome components and its final products exerted by Salmonella. Of note, IL18 protein levels in culture supernatants were negligible when measured by ELISA.
On the other hand, in vivo assays show completely different results. Firstly, B16F1 melanoma tumors express all inflammasome components including nlrp3, nlrc4, asc and procaspase-1. In addition, intratumoral administration of Salmonella to melanoma-bearing mice upregulates mRNA expression of nlrp3 and nlrc4 (but no changes were detected on the other two components analyzed) independently of flagellin presence. Moreover, we found upregulation of il1b but no il18 mRNA levels after Salmonella injection. We then analyzed IL18 protein levels in tumor homogenates, and we found no differences between groups (LVR01 or LVR01flag-, or untreated). However, it has been widely reported that melanoma per se produces and secretes large amounts of IL18 (Park, 2001), suggesting that these high baseline levels could be concealing a modest IL18 increase as a result of inflammasome activation mediated by Salmonella. For this reason we suggest that IL1β and caspase-1 protein determination would be determinant to demonstrate inflammasome activation. It is worth to mention that tumor outcome is also flagellin independent as mice treated with either LVR01 or LVR01flag-strains show comparative tumor volumes and survival rates, significantly better than untreated mice. Furthermore, we also found that proinflammatory cytokine and chemokine expression (il6, il12, cxcl1 and cxcl2) is significantly augmented by Salmonella injection in tumor homogenates, in a flagellin independent manner. Nonetheless we have not been able to identify the cell populations within upregulation is taking place because we studied total mRNA of tumor homogenates, without making any distinction between tumor cells and tumor-infiltrating immune cells.
Lastly, ongoing studies performed in nlrp3-/- and caspase-1-/- mice might help to finally unravel inflammasome role in Salmonella anti-tumoral effect exerted against subcutaneous melanoma.
We hope that this information helps us to elucidate the mechanisms by which Salmonella exerts its anti-tumoral effect, so as to design better strategies to address this worldwide affecting disease.
Acknowledgements
A. Mónaco was funded by a scholarship from the “Agencia Nacional de Investigación e Innovación” (ANII), Uruguay. Part of this project was financed by the "Comisón Sectorial de Investigación Científica" (CSIC), Uruguay.
References
Avilés, J. A., et al., Epidemiología y supervivencia del melanoma cutáneo en España: estudio de 552 casos (1994-2003). Revista Clínica Española, 2006. 206(7), 319–325.
Broz, P., et al., Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J Exp Med. 207(8): p. 1745-55.
Burdine, L., et al., Ipilimumab-induced colonic perforation. J Surg Case Rep. 2014(3).
Fellner, C., et al., Ipilimumab (yervoy) prolongs survival in advanced melanoma: serious side effects and a hefty price tag may limit its use. P T, 2012. 37(9): p. 503-30.
Franchi, L., et al., Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages. Nat Immunol, 2006. 7(6): p. 576-82.
Jack, A., et al., The treatment of melanoma with an emphasis on immunotherapeutic strategies. Surgical Oncology, 2006. 15(1), 13–24.
Hodi, F.S., et al., Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med, 2010. 363(8): p. 711-23.
Menzies, A.M., et al., New combinations and immunotherapies for melanoma: latest evidence and clinical utility. Ther Adv Med Oncol, 2013. 5(5): p. 278-85.
Miao, E.A., et al., Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat Immunol, 2006. 7(6): p. 569-75.
Raupach, B., et al., Caspase-1-mediated activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate immune defenses against Salmonella enterica serovar Typhimurium infection. Infect Immun, 2006. 74(8): p. 4922-6.
Shah, D.J. et al., Latest advances in chemotherapeutic, targeted, and immune approaches in the treatment of metastatic melanoma. Mayo Clin Proc, 2014. 89(4): p. 504-19.
Zeng, H., et al., Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J Immunol, 2003. 171(7): p. 3668-74.
Keywords:
Salmonella,
inflammasome modulation,
Flagellin,
melanoma -experimental-,
immunotherapy of cancer
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:
Poster Presentation
Citation:
Mónaco
A,
Yim
L,
Chabalgoity
JA and
Moreno
M
(2015). Salmonella, melanoma and inflammasome activation: unraveling networks.
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.2015.05.00143
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Received:
10 Jun 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Mrs. Amy Mónaco, Instituto de Higiene, Universidad de la República, Departamento de Desarrollo Biotecnológico, Montevideo, Estado…, 11200, Uruguay, amymonpat@gmail.com