Role of interplay between IL-4 and IFN-gamma in the in regulating M1 macrophage polarization induced by Nattectin

Recently our group described that Nattectin, a C-type lectin of the venom of Thalassophryne nattereri shows a potent pro-inflammatory capacity. Here, we demonstrated that Nattectin is able to induce M1 macrophage marker iNOS, and up-regulate the expression of MHC class II, CD80, CD86 and CD40 molecules. the increase in MHC class II and CD49a integrin expression with MMP-9 production and endocytic capacity depend on lectin function of Nattectin. Moreover, the polarization of peritoneal and bone marrow-derived macrophages induced by Nattectin to M1 profile is dependent on Th1 cytokines (IL-12 and IFN-gamma), and negatively regulated by Th2 cytokines (IL-4, IL-10 and IL-13). Also we reveal that IL-4 play a dual role in this polarization: a regular action of IL-4 was seen in the negative regulation of the CD40 expression, but an unexpected positive regulation was seen in the expression of CCR7 and MHC class II. Finally, our in vivo studies showed that the influx of neutrophils and small peritoneal macrophage - F4/80(low)MHCII(hi) induced by Nattectin is totally dependent on IL-4 and IFN-gamma cytokines. Furthermore, the induction of IL-6 release is negatively regulated by IL-4 and positively regulated by IL-12 and IFN-gamma. Together, the results allowed us to expand the knowledge about the regulation of macrophage activation, as well as confirmed the ability of Nattectin, a fish C-type lectin, as an important immunomodulatory agent. (c) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Inst Butantan, Unidade Imunorregulacao, Lab Especial Toxinol Aplicada, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Mol Immunol Innate Recognit Unit, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Mol Immunol Innate Recognit Unit, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

Immunomodulatory effect of the Tnp, a peptide isolated from the venom Thalassophyne nattereri on experimental autoimune encephalomyelitis.

Diante da ausência de tratamentos eficazes para a esclerose múltipla (EM) e sabendo que compostos animais têm sido usados como protótipos para o desenvolvimento de novas drogas, avaliamos o efeito do Tnp, um peptídeo cíclico inédito e com potencial antiinflamatório derivado do veneno de Thalassophryne nattereri, na encefalomielite autoimune experimental (EAE), um modelo representativo da EM. Demonstramos que o Tnp em distintos esquemas de tratamento por mecanismos também dependentes de IL-10 consegue diminuir a intensidade dos sintomas clínicos e adiar o pico de aparecimento dos sintomas graves na EAE por suprimir DC convencionais e propiciar DC plasmocitóides; por bloquear a infiltração de leucócitos e a reativação de clones Th1, Th17, microglia e macrófagos no SNC; por favorecer o aumento de células reguladoras e ainda por ultrapassar a BHE e alcançar o órgão alvo. O Tnp atenua a neuroinflamação e previne a desmielinização, refletindo assim na melhoria dos sinais clínicos na EAE, tornando-se um importante candidato para o tratamento da EM.Given the lack of effective treatments for multiple sclerosis (MS) and knowing that venomous have been used as prototype for the development of new drugs here we evaluated the effect of the Tnp, a described antiinflammatory cyclic peptide identified in the venom of Thalassophryne nattereri, on experimental autoimmune encephalomyelitis (EAE), a representative model of MS. We found that distinct treatments of Tnp by mechanisms also dependent on IL-10 significantly reduced the clinical severity of EAE. Tnp was related with: suppression of the activation state of conventional DC and the emergence of plasmacytoid DC; blocking the leukocyte infiltration and the reactivation of Th1, Th17, microglia cells and macrophages in the CNS; increasing of regulatory cells and also Tnp can overcome the BBB and reach the target organ. Tnp can reduce the severity of symptoms and delay the peak of onset of severe symptoms. These results shed light on the role of Tnp a small molecule in the regulation of inflammation and provides a new therapeutic opportunity for the treatment of MS

Role of innate TLR receptors in formation of humoral memory and long-life lymphocytes B: action of natterins proteases, majority toxins of Thalassophryne nattereri venom.

A contribuição de células B para a memória imunológica se dá por duas distintas populações: células B de memória e células produtoras de anticorpos de longa vida (ASC). A inter-relação entre estas células bem como os mecanismos envolvidos para a manutenção destas tem sido pouco entendida. O veneno de Thalassophryne nattereri tem se mostrado capaz de induzir uma intensa resposta imune de memória. Nós avaliamos o efeito das Natterinas, que são as toxinas majoritárias e inéditas, na indução e manutenção da resposta imune de memória de células B. Este estudo, além de permitir um maior esclarecimento da resposta humoral de memória induzida pelo veneno do peixe T. nattereri, permitiu o estudo da complexa organização do compartimento de células B de memória e ASCs. Também evidenciamos a importância da atividade proteásica para a manutenção da cronicidade de resposta de células B no peritônio, no baço e na medula, como verificamos que a ativação de receptores inatos como osTLRs é decisiva para a geração e manutenção de ASCs B220pos/neg em resposta às Natterinas, dependentes das vias de sinalização MyD88 ou TRIF. Estas sinalizações controlam a magnitude, a qualidade e a longa duração da resposta humoral de memória.The contribution of B cells for the immunological memory feels for two different populations: memory B cells and long-lived antibodies secreting cells (ASC). The interrelation among these cells as well as the mechanisms involved for the maintenance of these it has been little understood. The venom of Thalassophryne nattereri possesses the ability to induce an intense memory immune response. We evaluated the effect of Natterins that are majority toxins in the venom, in the induction and maintenance of the immune memory response of cells B. The study, besides allowing a larger explanation of the humoral memory response induced by the venom of the fish, it allowed the understanding of the complex organization of the memory B cells compartment, mainly of the subtype of long-lived cells (ASC). Also, we showed the importance of the protease activity of Natterins in the maintenance of the chronic B cell responses in the three analyzed compartments. We verify that the activation of Toll like receptors is decisive for the generation and maintenance of ASCs B220pos/neg in response to Natterins, dependent on the MyD88 or TRIF signaling that control the quality and the duration of the humoral memory response

The Longevity of Th2 Humoral Response Induced by Proteases Natterins Requires the Participation of Long-Lasting Innate-Like B Cells and Plasma Cells in Spleen

<div><p>The generation of long-lived antibody-secreting cells (ASC) and memory B cells are critical events for an effective vaccine and the choice of adjuvant can influence these processes. Various cellular and molecular mechanism involved in the protease action that determine Th2 responses have been identified. However, direct or indirect actions in the regulation of the induction, survival and longevity of ASC in differential compartments remain largely unknown. We investigated whether the proteolytic activity of proteins are determinant for the modulation of the memory immune response in mice, promoting the differentiation of memory B cells to terminally differentiated end stage cells. Here, we show that the proteolytic activity of Natterins, from the venom of <i>Thalassophryne nattereri</i> Brazilian fish, besides inducing a Th2 response with plasmatic titers of high-affinity antigen-specific IgE over extended periods is sufficient for the generation of signals that contribute to the formation of a survival niche in the spleen, essential for the longevity of the main subtype of ASC with B220^neg phenotype.</p></div

TLR2, TLR4 and the MyD88 Signaling Are Crucial For the <i>In Vivo</i> Generation and the Longevity of Long-Lived Antibody-Secreting Cells

<div><p>This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our <i>in vivo</i> findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220^pos. TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220^pos (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220^neg in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220^pos (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220^pos (MyD88-independent) and ASC B220^neg into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220^neg in peritoneum and BM. Terminally differentiated ASC B220^neg required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220^pos rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220^pos and ASC B220^neg.</p></div

The importance of the integration of signaling pathways downstream of SP1R and TLRs in modulating the maintenance of ASC in peritoneal cavity.

<p>We propose that during secondary immune response against Natterins, after some period in the lymphoid organs, S1PRs are required for TLR-induced peritoneal B1a and B2 cell egress and also for medullar B2 longevity; and that the longevity of Bmem in splenic niche, the intensity of ASC B220^pos into peritoneal cavity and BM, and mainly the longevity of ASC B220^neg into inflamed peritoneal tissue is strong supported by CXCR4 expression dependent on SP1R expression in B lymphocytes and its recirculation through lymphoid organs.</p

The impaired lymphocyte egress from the secondary lymph organs controls the migration of innate-like B cell and memory B cell for peripheral tissues.

<p>Cells from peritoneum, spleen and bone marrow of Natterins-immunized BALB/c mice treated or not with FTY720 were obtained at day 28. The bars representative of the absolute numbers of B1a cells (B220⁺CD5⁺) (<b><i>A</i></b>), B1b cells (B220^lowCD5^neg) (<b><i>B</i></b>), B2 cells (B220+CD23+) (<b><i>C</i></b>), Bmem (B220⁺CD19⁺IgG⁺) (<b><i>D</i></b>) ASC B220^pos (CD138⁺CD43⁺B220⁺) (<b><i>E</i></b>), ASC B220^neg (CD138⁺CD43⁺B220^neg) (<b><i>F</i></b>) were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD5, Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220, and Rat IgG2ak PE-anti-mouse CD23, Armenian hamster IgG1y2 FITC-anti-mouse CD19, Goat IgG2bk PE-anti-mouse IgG (specific for IgG1, IgG2a, IgG2b and IgG3), Rat IgG2ak FITC-anti-mouse CD43, and Rat IgG2ak PE-anti-mouse CD138. *<i>p</i><0.05 compared to control mice; and ^#<i>p</i><0.05 compared to Natterins-immunized mice without treatment.</p

TLR4 is important for the transient expansion of B2 cells in bone marrow, but TLR2 and MyD88 are crucial for their survival in this niche.

<p>A representative dot plot of B2 cells (B220^highCD23^pos) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained from TLR4 mutant (<i>left</i>) or from TLR2 <i>KO</i> and MyD88 <i>KO</i> (<i>right</i>) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220^highCD23^pos cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD23, Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220. *<i>p</i><0.05 compared to control mice; and ^#<i>p</i><0.05 compared to <i>WT</i> mice immunized with Natterins.</p

The persistence of B1b cells in bone marrow is negatively regulated by TLR2/TLR4 and MyD88 signals.

<p>A representative dot plot of B1b cells (B220^lowCD5^neg) analyses is shown. Cells from peritoneum (A, D), spleen (B, E) and bone marrow (C, F) were obtained from TLR4 mutant (<i>left</i>) or from TLR2 <i>KO</i> and MyD88 <i>KO</i> (<i>right</i>) Natterins immunized mice after 48, 74 and 120 days. The bars representative of the absolute numbers of B220^lowCD5^neg cells were determined from total mononuclear cells by multiparametric flow cytometer using Rat IgG2ak PE-anti-mouse CD5, and Rat IgG2ak PerCP-Cy5-anti-mouse CD45R/B220. *<i>p</i><0.05 compared to control mice; and ^#<i>p</i><0.05 compared to <i>WT</i> mice immunized with Natterins.</p

Possible scenario for the effect of Natterins on differentiation and survival of ASC in spleen.

<p>The development of long-term immunity to Natterins could be characterized by persistent anaphylactic-Abs derived from continuous differentiation in germinal center of Bmem from innate-like B1b and B2 cells and derived from linear differentiation of ASC B220^neg from B220^pos and Bmem.</p