IL-33 receptor (ST2) deficiency downregulates myeloid precursors, inflammatory NK and dendritic cells in early phase of sepsis.

BACKGROUND: Sepsis is a life-threatening disease mediated by profound disturbances in systemic inflammatory response to infection. IL-33 is multifunctional regulator of numerous aspects of innate and adaptive immune response. The aim of this article was to further evaluate the role of IL-33 receptor (ST2) in different pathways of innate immunity during early polymicrobial sepsis. METHODS: Polymicrobial sepsis was induced using cecal ligation and puncture (CLP) model in ST2 deficient (ST2-/-) and wild type BALB/c mice. Peritoneal and spleen cells were isolated for further phenotyping. Apoptosis was determined by immunohistochemistry and flow cytometry. RESULTS: Deletion of ST2 leads to increased susceptibility to early manifestations of sepsis as evaluated by clinical signs and survival. These are accompanied by decrease in the total number of neutrophils, eosinophils and mast cells in peritoneal cavity 12 h after CLP. In early sepsis there was also low number of precursors of myeloid cells in particular CD11b+Ly6G+Ly6Clow cells in spleen of ST2-/- mice. Although the number of NK cells in the spleen was similar, there were significant differences in the presence of inflammatory IFN-γ and IL-17 producing NK cells. Further, ST2 deletion affects the phenotype and maturation of dendritic cell in sepsis. The total number of dendritic cells in the spleen was lower as well as IL-12 expressing dendritic cells. Finally, there was higher frequency of active caspase-3 positive and early apoptotic cells, in particular CD11c positive cells, in spleen of septic ST2-/- mice. CONCLUSION: Taken together, our data provide the evidence that ST2 deficiency in early phase of sepsis downregulates myeloid precursors, inflammatory NK and dendritic cells.

The Potential of Frog Skin-Derived Peptides for Development into Therapeutically-Valuable Immunomodulatory Agents.

The aim of this article is to review the immunoregulatory actions of frog skin-derived peptides in order to assess their potential as candidates for immunomodulatory or anti-inflammatory therapy. Frog skin peptides with demonstrable immunomodulatory properties have been isolated from skin secretions of a range of species belonging to the families Alytidae, Ascaphidae, Discoglossidae, Leptodactylidae, Pipidae and Ranidae. Their effects upon production of inflammatory and immunoregulatory cytokines by target cells have been evaluated ex vivo and effects upon cytokine expression and immune cell activity have been studied in vivo by flow cytometry after injection into mice. The naturally-occurring peptides and/or their synthetic analogues show complex and variable actions on the production of proinflammatory (TNF-α, IL-1ß, IL-12, IL-23, IL-8, IFN-γ and IL-17), pleiotropic (IL-4 and IL-6) and immunosuppressive (IL-10 and TGF-ß) cytokines by peripheral and spleen cells, peritoneal cells and/or isolated macrophages. The effects of frenatin 2.1S include enhancement of the activation state and homing capacity of Th1-type lymphocytes and NK cells in the mouse peritoneal cavity, as well as the promotion of their tumoricidal capacities. Overall, the diverse effects of frog skin-derived peptides on the immune system indicate their potential for development into therapeutic agents.

The frog skin host-defense peptide frenatin 2.1S enhances recruitment, activation and tumoricidal capacity of NK cells.

Frog skin is a source of peptides with various biological properties. Frenatin 2.1S, derived from norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus, exhibits immunostimulatory effects as demonstrated by the promotion of proinflammatory phenotypes of mononuclear cells in mouse peritoneal cavity and spleen. The aim of this study was to identify the populations of host cells sensitive to the action of frenatin 2.1S in vivo and to study its effects on their functional antitumor capacity. A single injection of frenatin 2.1S (100µg) in BALB/c mice increased the presence of peritoneal CD11c+ dendritic cells and CD3+ T cells 24h after administration and there was a significant increase in the number of IL-17 and CXCR3 expressing inflammatory T cells. Frenatin 2.1S treatment also increased the number of TNF-α expressing F4/80+ proinflammatory M1 macrophages. The most striking finding of the study is the marked increase of the number of peritoneal natural killer (NK) cells following frenatin 2.1S injection. Further, frenatin 2.1S administration led to activation of NK cells as evaluated by increased expression of NKG2D, FasL, CD69 and CD107a. The increased ratio of interferon-γ vs. IL-10 producing NK cells is further indication of the proinflammatory action of frenatin 2.1S. Peptide treatment enhanced the tumoricidal action of peritoneal NK cells on 4T1 mouse mammary carcinoma cells as revealed by the real-time automated monitoring of cell status. Our data demonstrate that frenatin 2.1S promotes activation and cytotoxic capacity of NK cells and should be regarded as a candidate for antitumor immunotherapy.

In vivo administration of the frog skin peptide frenatin 2.1S induces immunostimulatory phenotypes of mouse mononuclear cells.

Host-defense peptides secreted by epithelial cells exhibit cytotoxic and immunoregulatory effects in order to protect the organism against invading microorganisms. Antimicrobial peptides derived from frog skin display both immunostimulatory and immunosuppressive actions as demonstrated by in vitro cytokine production by macrophages. Frenatin 2.1S, first isolated from skin secretions of the frog, Sphaenorhynchus lacteus (Hylidae), enhances the in vitro production of pro-inflammatory IL-1ß, TNF-α and IL-23 by mouse peritoneal cells. In order to test whether the immunostimulatory action of frenatin 2.1S may be reproduced in vivo, effects of intraperitoneal injections of this peptide on mononuclear cells in the peritoneum and spleen were determined 24h after administration. The data indicate that frenatin 2.1S enhances the activation state and homing capacity of Th1 type lymphocytes and NKT cells in the mouse peritoneal cavity, as evaluated by increased expression of early activation marker CD69 among T and NKT cells and chemokine receptor CXCR3 among T cells. Frenatin 2.1S significantly increases the percentage of (F4/80(+)CD11c(+)CD206(+)) pro-inflammatory M1 macrophages and enhances the expression of MHC class II molecules on F4/80(+)CD11c(+) macrophages in the mouse peritoneal cavity. Additionally, injection of frenatin 2.1S, in the presence or absence of lipopolysaccharide, increases the percentage of peritoneal B cells of the (CD19(+)CD11b(+)CD5(+)) B1a phenotype thus contributing to an inflammatory milieu. We suggest that the immunostimulatory effect of frenatin 2.1S may have therapeutic relevance in disease states, such as certain types of cancer, in which an enhanced inflammatory response may be beneficial.

Effects of tigerinin peptides on cytokine production by mouse peritoneal macrophages and spleen cells and by human peripheral blood mononuclear cells.

The tigerinins are a family of cationic, cyclic peptides of unknown biological function produced in the skins of diverse frog species. Tigerinin-1R (RVCSAIPLPICH.NH2) from Hoplobatrachus rugulosus (Dicroglossidae), tigerinin-1V (RICYAMWIPYPC) from Lithobates vaillanti (Ranidae), and tigerinin-1M (WCPPMIPLCSRF.NH2) from Xenopus muelleri (Pipidae) did not inhibit growth of Escherichia coli and Staphylococcus aureus at concentrations up to 500 µg/ml and were not hemolytic. Incubation of peritoneal macrophages from both BALB/c and C57BL/6 mice with tigerinin-1M, -1R and -1V (20 µg/ml) significantly (P < 0.05) increased production of the anti-inflammatory cytokine IL-10 and potentiated the stimulation produced by lipopolysaccharide (LPS). Incubation with the tigerinins (20 µg/ml) significantly increased production of IL-6 in LPS-stimulated macrophages from C57BL/6 mice but only tigerinin-1V potentiated IL-6 production in LPS-stimulated macrophages from BALB/c mice. The tigerinins did not have significant effects on the production of proinflammatory cytokines IL-12 and IL-23 by macrophages from BALB/c mice. In a population of mononuclear cells derived from mouse spleen, tigerinin-1M and -1V suppressed production of IFN-γ with no effect on IL-17 production and the three tigerinins enhanced IL-10 production. The three tigerinins (≤ 5 µg/ml) also significantly increased production of IL-10 in unstimulated and LPS-stimulated human peripheral blood mononuclear cells. The data indicate that the tigerinins may function as immunomodulatory host-defense peptides in frog skin.

Interleukin-33/ST2 axis promotes breast cancer growth and metastases by facilitating intratumoral accumulation of immunosuppressive and innate lymphoid cells.

The role of IL-33/ST2 pathway in antitumor immunity is unclear. Using 4T1 breast cancer model we demonstrate time-dependent increase of endogenous IL-33 at both the mRNA and protein levels in primary tumors and metastatic lungs during cancer progression. Administration of IL-33 accelerated tumor growth and development of lung and liver metastases, which was associated with increased intratumoral accumulation of CD11b(+) Gr-1(+) TGF-ß1(+) myeloid-derived suppressor cells (MDSCs) that expressed IL-13α1R, IL-13-producing Lin(-) Sca-1(+) ST2(+) innate lymphoid cells (ILCs) and CD4(+) Foxp3(+) ST2(+) IL-10(+) Tregs compared to untreated mice. Higher incidence of monocytic vs. granulocytic MDSCs and plasmocytoid vs. conventional dendritic cells (DCs) was present in mammary tumors of IL-33-treated mice. Intratumoral NKp46(+) NKG2D(+) and NKp46(+) FasL(+) cells were markedly reduced after IL-33 treatment, while phosphate-buffered saline-treated ST2-deficient mice had increased frequencies of these tumoricidal natural killer (NK) cells compared to untreated wild-type mice. IL-33 promoted intratumoral cell proliferation and neovascularization, which was attenuated in the absence of ST2. Tumor-bearing mice given IL-33 had increased percentages of splenic MDSCs, Lin(-) Sca-1(+) ILCs, IL-10-expressing CD11c(+) DCs and alternatively activated M2 macrophages and higher circulating levels of IL-10 and IL-13. A significantly reduced NK cell, but not CD8(+) T-cell cytotoxicity in IL-33-treated mice was observed and the mammary tumor progression was not affected when CD8(+) T cells were in vivo depleted. We show a previously unrecognized role for IL-33 in promoting breast cancer progression through increased intratumoral accumulation of immunosuppressive cells and by diminishing innate antitumor immunity. Therefore, IL-33 may be considered as an important mediator in the regulation of breast cancer progression.

Conformational analysis of the frog skin peptide, plasticin-L1, and its effects on production of proinflammatory cytokines by macrophages.

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance/molecular dynamics characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 µg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1ß, IL-12, IL-23, and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, whereas the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Galectin-3 is a regulator of metaflammation in adipose tissue and pancreatic islets.

The cells of the innate and adaptive immune systems have been implicated in the development of obesity-induced metaflammation and metabolic disorders including type 2 diabetes. Galectin-3, a ß-galactoside-binding lectin, modulates immune/inflammatory responses and specifically binds to advanced glycation end products (AGE), modified lipoproteins, and endotoxin. In the recently published study we demonstrate proinflammatory changes in the visceral adipose tissue and pancreatic islets in galectin-3-deficient mice fed high-fat diet which also exhibited excess adiposity, hyperglycemia, insulin resistance and systemic inflammation compared with their diet matched wild-type controls. This was associated with the increased incidence of Type-1 T and NKT cells and pro-inflammatory CD11c(+)CD11b(+) macrophages in the visceral adipose tissue. Severe insulitis, infiltration of macrophages expressing NLRP3 inflammasome and IL-1ß, and enhanced accumulation of AGE were present within the pancreatic islets in obese LGALS3(-/-) mice. Moreover, increased caspase-1 dependent IL-1ß secretion with increased expression of NLRP3 inflammasome and phospho-NFκBp65 were observed in LGALS3(-/-) peritoneal macrophages stimulated in vitro by lipopolysaccharide and/or saturated fatty acid palmitate. The amplified high-fat diet-induced obesity and hyperglycemia and exacerbated inflammation in adipose tissue and pancreatic islets in LGALS3(-/-) mice suggest an important role for galectin-3 in the regulation of adiposity, metaflammation and type 2 diabetes.

Galectin-3 deficiency accelerates high-fat diet-induced obesity and amplifies inflammation in adipose tissue and pancreatic islets.

Obesity-induced diabetes is associated with low-grade inflammation in adipose tissue and macrophage infiltration of islets. We show that ablation of galectin-3 (Gal-3), a galactoside-binding lectin, accelerates high-fat diet-induced obesity and diabetes. Obese LGALS3(-/-) mice have increased body weight, amount of total visceral adipose tissue (VAT), fasting blood glucose and insulin levels, homeostasis model assessment of insulin resistance, and markers of systemic inflammation compared with diet-matched wild-type (WT) animals. VAT of obese LGALS3(-/-) mice exhibited increased incidence of type 1 T and NKT lymphocytes and proinflammatory CD11c(+)CD11b(+) macrophages and decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells and M2 macrophages. Pronounced mononuclear cell infiltrate, increased expression of NLRP3 inflammasome and interleukin-1ß (IL-1ß) in macrophages, and increased accumulation of advanced glycation end products (AGEs) and receptor for AGE (RAGE) expression were present in pancreatic islets of obese LGALS3(-/-) animals accompanied with elevated phosphorylated nuclear factor-κB (NF-κB) p65 and mature caspase-1 protein expression in pancreatic tissue and VAT. In vitro stimulation of LGALS3(-/-) peritoneal macrophages with lipopolysaccharide (LPS) and saturated fatty acid palmitate caused increased caspase-1-dependent IL-1ß production and increased phosphorylation of NF-κB p65 compared with WT cells. Transfection of LGALS3(-/-) macrophages with NLRP3 small interfering RNA attenuated IL-1ß production in response to palmitate and LPS plus palmitate. Obtained results suggest important protective roles for Gal-3 in obesity-induced inflammation and diabetes.

An immunomodulatory peptide related to frenatin 2 from skin secretions of the Tyrrhenian painted frog Discoglossus sardus (Alytidae).

Norepinephrine-stimulated skin secretions of the Tyrrhenian painted frog Discoglossus sardus Tschudi, 1837 (Alytidae) did not contain any peptide with antimicrobial or hemolytic activity. However, peptidomic analysis of the secretions revealed the presence of an abundant peptide with structural similarity to frenatin 2, previously isolated from the Australian frog Litoria infrafrenata (Hylidae). The primary structure of the peptide, termed frenatin 2D, was established as DLLGTLGNLPLPFI.NH2 by automated Edman degradation and mass spectrometry with electron-transfer dissociation (ETD)-based fragmentation and confirmed by chemical synthesis. The structure of a second frenatin 2-related peptide, termed frenatin 2.1D, that was present in much lower abundance was established as GTLGNLPAPFPG. Frenatin 2D (20 µg/ml) significantly stimulated production of the proinflammatory cytokines TNF-α (P<0.05) and IL-1ß (P<0.01) by mouse peritoneal macrophages but the peptide did not potentiate the stimulation produced by lipopolysaccharide (LPS). The peptide increased IL-12 production in both unstimulated (P<0.01) and LPS-stimulated (P<0.05) cells but stimulatory effects on IL-6 production were not significant. The biological role of frenatin 2D is unknown but it is speculated that the peptide acts on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms.