The axis IL-10/claudin-10 is implicated in the modulation of aggressiveness of melanoma cells by B-1 lymphocytes

B-1 lymphocytes are known to increase the metastatic potential of B16F10 melanoma cells via the extracellular signal-regulated kinase (ERK) pathway. Since IL-10 is associated with B-1 cells performance, we hypothesized that IL-10 could be implicated in the progression of melanoma. In the present work, we found that the C57BL/6 mice, inoculated with B16F10 cells that were co-cultivated with B-1 lymphocytes from IL-10 knockout mice, developed fewer metastatic nodules than the ones which were injected with the melanoma cells that were cultivated in the presence of wild-type B-1 cells. The impairment of metastatic potential of the B16F10 cells was correlated with low activation of the ERK signaling pathway, supporting the idea that the production of IL-10 by B-1 cells influences the behavior of the tumor. A microarray analysis of the B-1 lymphocytes revealed that IL-10 deficiency is associated with down-regulation of the genes that code for claudin-10, a protein that is involved in cell-to-cell contact and that has been linked to lung adenocarcinoma. In order to determine the impact of claudin-10 in the cross-talk between B-1 lymphocytes and the B16F10 tumor cells, we took advantage of small interfering RNA. The silencing of claudin-10 gene in B-1 lymphocytes inhibited activation of the ERK pathway and abrogated the B-1-induced aggressive behavior of the B16F10 cells. Thus, our findings suggest that the axis IL-10/claudin-10 is a promising target for the development of therapeutic agents against aggressive melanoma.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Univ Paulista, Environm & Expt Pathol Program, Sao Paulo, SP, BrazilUniv Fed Sao Paulo Escola Paulista Med UNIFESP EPM, Dept Microbiol Immunol & Parasitol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Pharmaceut Sci, Campus Diadema, Diadema, SP, BrazilCtr Univ Sao Camilo, Sao Paulo, SP, BrazilUniv Fed Sao Paulo Escola Paulista Med UNIFESP EPM, Nephrol Div, Dept Med, Sao Paulo, SP, BrazilUniv Fed Sao Paulo Escola Paulista Med UNIFESP EPM, Dept Microbiol Immunol & Parasitol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Pharmaceut Sci, Campus Diadema, Diadema, SP, BrazilUniv Fed Sao Paulo Escola Paulista Med UNIFESP EPM, Nephrol Div, Dept Med, Sao Paulo, SP, BrazilFAPESP: 11/50256-6FAPESP: 08/50632-5FAPESP: 2016/02189-1CNPq: 472035/2011-8CNPq: 308374/2016-9Web of Scienc

B16 melanoma cells increase B-1 cell survival, IL-10 production and radioresistance in vitro

B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. the role of both cell populations in cancer progression is still controversial. Previous studies have indicated that direct contact between B-1 cells and B16 melanoma tumor cells (B16) increases the metastatic potential of the tumor cells. However, cellular changes that are induced in B-1 cells during the interaction between these two cell types have not been evaluated. in the present study, it is hypothesized that B-1 cells are modified after their interaction with tumor cells, leading to both increased cell viability and rate of proliferation. Additionally, soluble factors that were secreted by B16 cells were sufficient to augment B-1 cell viability and to modify the production of IL-10 by B-1 cells. Impressively, after direct or indirect contact with the B16 cells, B-1 cells became resistant to radiation-induced cell death. Thus, future studies that assess the importance of concomitant immunity and other conventional therapies in cancer treatment are needed. (C) 2012 Elsevier GmbH. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de PesquisaCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Discipline Immunol, BR-04023900 São Paulo, BrazilUniv Paulista UNIP, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Discipline Immunol, BR-04023900 São Paulo, BrazilFAPESP: 2008/58561-0Conselho Nacional de Pesquisa: CNPq - 142676/07-1CAPES: 1326/09-0Web of Scienc

Cross Talk between Peritoneal Macrophages and B-1 Cells <i>In Vitro</i>

<div><p>B-1 cells constitute a distinct B cell population with unique phenotypic and functional characteristics. They represent the main B cell population found in mouse peritoneal and pleural cavities. The communication between B-1 cells and peritoneal macrophages has been previously studied, and the effect this interaction has on macrophages has been previously described. Using an <i>in vitro</i> co-culture model, herein we demonstrated that peritoneal macrophages were able to increase survival rates and to stimulate proliferation of B-1 cells. IL-6 was also found to be important in B-1 cell survival; recombinant IL-6 increases the percentage of viable B-1 cells in culture. Furthermore, molecules involved in the IL-6 signaling pathway, such as STAT-3 and Bcl-2, were highly expressed in B-1 cells after co-culture with peritoneal macrophages. IL-6-deficient peritoneal macrophages were not able to increase B-1 cell survival, confirming the importance of this cytokine. Altogether, our results indicate a novel mechanism in which peritoneal macrophages are able to regulate the B-1 population via IL-6 secretion.</p></div

IL-6 cytokine increases pSTAT-3 and Bcl-2 expression.

<p>(A) Representative western blot of STAT-3, pSTAT-3 and Bcl-2 of B-1 cells cultivated alone (line 1), with BALB/c peritoneal macrophages (line 2), with BALB/c conditioned medium (line 3) or with 800 pg/ml of recombinant IL-6 (line 4), after 24 hours. Results are representative of 3 independent experiments. Relative expression of total (B) STAT-3, (C) pSTAT-3 and (D) Bcl-2 compared to β-actin are shown in the graphs. *p<0.05, **p<0.01, ***p<0.001.</p

Peritoneal macrophages derived from IL-6 KO mouse are unable to maintain B-1 cells viability.

<p>B-1 cells derived from C57BL/6 wild type or IL-6 KO mouse were cultivated with wild type or IL-6 KO peritoneal macrophages (A), or with conditioned medium derived from IL-6 KO or WT mouse (B), and viability was measured after 24 hours. (C) IL-6 was measured when B-1 cells derived from wild type or IL-6 KO mouse were cultivated with wild type or IL-6 KO peritoneal macrophages. (D) Anti IL-6 was added to the culture, and B-1 cells viability was measured. *p<0.05, **p<0.01, ***p<0.001.</p

BALB/<i>Xid</i> peritoneal macrophages and conditioned medium augment B-1 cell survival.

<p>(A) Representative dot plot of BALB/<i>Xid</i> peritoneal macrophages stained with F4/80, CD11b (left panel) and CD19 (right panel). (B) Graph represents percentage of viable B-1 cells present in culture after 24 and 72 hours when B-1 cells were cultivated alone, on BALB/c or BALB/<i>Xid</i> peritoneal macrophages or with BALB/c or BALB/<i>Xid</i> conditioned medium. ***p<0.001.</p

IL-6 increases B-1 cell viability.

<p>(A) IL-6 and IL-10 (B) were measured when B-1 cells were cultivated alone or with BALB/c or BALB/<i>Xid</i> conditioned medium, after 24 and 72 hours. (B) B-1 cells were cultivated alone or with 200, 400 or 800 pg/ml of recombinant IL-6, and their survival was measured. *p<0.05, **p<0.01, ***p<0.001.</p

A soluble factor is essential for B-1 cell survival <i>in vitro</i>.

<p>(A) Graph represents percentage of viable B-1 cells when cultivated alone, together with peritoneal macrophages or with conditioned medium, after 24 and 72 hours. (B) Viability was measured when B-1 cells were cultivated with different concentration of the conditioned medium. (C) Survival of B-1 cells was evaluated when they were cultured on transwells placed on peritoneal macrophages. *p<0.05, **p<0.01, ***p<0.001.</p