IL-12p70 Production Research Articles

Modulation of functional activity of heat-stressed chicken macrophages by poultry-derived probiotic lactobacilli

This study investigated the potential role of lactobacilli in mitigating the negative effects of heat stress on the functional activity of chicken macrophages. Macrophage-like MQ-NCSU cells were incubated at 40 and 44 °C in the presence or absence of a single or a mixture of different poultry-derived Lactobacillus spp., including Lactobacillus animalis, L. acidophilus, L. reuteri, and L. crispatus. Macrophage activation was evaluated by measuring nitric oxide (NO) production, phagocytic activity, and the transcription levels of cytokines, chemokine, and Toll-like receptors (TLRs). Macrophages exposed to heat stress exhibited increased production of NO, diminished expression of interleukin (IL)-1β and IL-12p40, and elevated expression of TLR2 and TLR4, whereas no significant alterations in the phagocytic activity of macrophages were observed. Conversely, treatment of macrophages with probiotic lactobacilli counteracted the effects associated with heat stress. This was evidenced by a notable enhancement in macrophage phagocytic activity, NO production and expression of IL-1β, IL-12p40, IL-18, and chemokine CXCL8, coupled with a reduction in TLR2 and TLR4 expression. These findings suggest that probiotic lactobacilli could be given to chickens to mitigate the negative effects of heat stress on their innate immune system. However, further studies are required to validate the observed effects in an in vivo model.

Combined TLR-3/TLR-8 Signaling in the Presence of α-Type-1 Cytokines Represents a Novel and Potent Dendritic Cell Type-1, Anti-Cancer Maturation Protocol.

During the ex vivo generation of anti-cancer dendritic cell (DC)-based vaccines, their maturation still represents one of the most crucial steps of the manufacturing process. A superior DC vaccine should: possess extensive expression of co-stimulatory molecules, have an exceptional type-1 polarization capacity characterized by their ability to produce IL-12p70 upon contact with responding T cells, migrate efficiently toward chemokine receptor 7 (CCR7) ligands, and have a superior capacity to activate cytotoxic T cell responses. A major advance has been achieved with the discovery of the next generation maturation protocol involving TLR-3 agonist (poly I:C), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IFN-α, and has since been known as α-type-1 maturation cocktail. We demonstrate how this combination can be greatly enhanced by the inclusion of a TLR-8 stimulation (R848), thereby contributing to potentiation between different TLR signaling pathways. For maximum efficiency, TLR-3 stimulation should precede (termed pre I:C) the stimulation with the R848/TNF-α/IL-1β/IFN-α/IFN-γ cocktail. When compared to DCs matured with α-type-1 maturation cocktail (αDCs), DCs matured with pre I:C/R848/TNF-α/IL-1β/IFN-α/IFN-γ (termed zDCs) displayed higher expression of CD80 and CD86 co-stimulatory molecules. Importantly, after CD40-ligand stimulation, which simulates DC-T cell contact, zDCs were much more proficient in IL-12p70 production. In comparison to αDCs, zDCs also displayed a significantly greater migratory capacity toward chemokine ligands (CCL)19 and CCL21, and had a significantly greater allo-stimulatory capacity. Finally, zDCs were also superior in their capacity to induce melanoma-specific CD8+ T cells, CD8+ T cell proliferation, and cytotoxic T cells, which produced approximately two times more IFN-γ and more granzyme B, than those stimulated with αDCs. In conclusion, we present a novel and superior DC maturation cocktail that could be easily implemented into next generation DC vaccine manufacturing protocols in future trials.

Adipose invariant NKT cells interact with CD1d-expressing macrophages to regulate obesity-related inflammation.

Obesity is accompanied by and accelerated with chronic inflammation in adipose tissue, especially visceral adipose tissue (VAT). This low-level inflammation predisposes the host to the development of metabolic disease, most notably type 2 diabetes. We have focused on the capacity of glycolipid-reactive, CD1d-restricted natural killer T (NKT) cells to modulate obesity and its associated metabolic sequelae. We previously reported that CD1d knockout (KO) mice are partially protected against the development of obesity-associated insulin resistance, and these findings were recapitulated in mice with an adipocyte-specific CD1d deficiency, suggesting that NKT cell-adipocyte interactions play a critical role in exacerbating disease. However, many other CD1d-expressing cells contribute to the in vivo responses of NKT cells to lipid antigens. In the present study, we examined the role of CD1d expression by macrophages (Mϕ) in the development of obesity-associated metabolic inflammation using LysMcre-cd1d1f/f mice where the CD1d1gene is disrupted in a Mϕ-specific manner. Unexpectedly, these animals contained a higher frequency of T-bet+ CD4+ T cells in VAT with increased production of Th1 cytokines that aggravated VAT inflammation. Mϕ from mutant mice displayed increased production of IL-12p40, suggesting M1 polarization. These findings indicate that interactions of CD1d on Mϕ with NKT cells play a beneficial role in obesity-associated VAT inflammation and insulin resistance with a sharp contrast to an aggravating role of CD1d in another type of antigen-presenting cell, dendritic cells.

Glutamate in the medium of Lactiplantibacillus plantarum FL-664 affects the production of IL-12(p40) on murine spleen cells.

Lactic acid bacteria (LAB) have been attracting attention for their effects on innate immunity, and therefore, it is required to develop an efficient culturing method while maintaining their functionality. In this study, first, we compared the growth and functionality of LAB cultured on food grade (FG) medium with those on standard LAB medium and found that LAB cultured in the FG medium were smaller in cell size with high yield and had a higher ability to induce IL-12(p40) production by murine spleen cells in vitro. Moreover, the higher the glutamate concentration in the medium, the smaller the cell size, and the higher the yield and the higher the ability to induce IL-12 production. Addition of glutamate to the culture medium changes the size of LAB and affects their ability to induce IL-12(p40) production. In conclusion, regulating the concentration of glutamate would be important in the efficient culturing of functional LAB.

The effects of 1,25(OH)2 D3 treatment on immune responses and intracellular metabolic pathways of bone marrow-derived dendritic cells from lean and obese mice.

Vitamin D affects differentiation, maturation, and activation of dendritic cells (DCs). Obesity-related immune dysfunction is associated with metabolic changes in immune cells. Objectives of the study are to investigate the effects of vitamin D and obesity on immune responses and markers related to immunometabolism of bone marrow-derived dendritic cells (BMDCs). Bone marrow cells (BMCs) were isolated from lean and obese mice, and BMDCs were generated by culturing BMCs with rmGM-CSF. BMDCs were treated with 1 or 10nM of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), and maturation was induced by LPS (50 ng/ml) stimulation for 24 hr. Cell phenotypes, cytokine productions, and expression of proteins and genes involved in Akt/mTOR signaling pathway and glycolytic pathway were determined. 1,25(OH)2 D3 treatment inhibited differentiation of BMDCs (CD11c+ %), expression of phenotypes related with DC function (MHC class II and CD86) and production of IL-12p70 in both lean and obese mice. The expression of PD-L1 and the ratio of IL-10/IL-12p70 were increased by 1,25(OH)2 D3 . With 1,25(OH)2 D3 treatment, Akt/mTOR signaling pathway was suppressed, and expression of genes related to glycolysis (Glut1, Pfkfb4, and Hif1A) was increased. The upregulation of glycolysis-related genes observed with 1,25(OH)2 D3 treatment seems to be associated with the induction of tolerogenic features of BMDCs from lean and obese mice, and Hif1A seems to have a potential role in conveying the effect of 1,25(OH)2 D3 on glycolysis.

Immunogenic profiling of Mycobacterium tuberculosis DosR protein Rv0569 reveals its ability to switch on Th1 based immunity

Mycobacterium tuberculosis (M.tb) is a multifaceted bacterial pathogen known to infect more than 2 billion people globally. However, a majority of the individuals (>90%) show no overt clinical symptoms of active Tuberculosis (TB) and, it is reported that M.tb in these individuals resides in the latent form. Therefore, a huge burden of latently infected population poses serious threat to the human health. Inconsistent efficacy of BCG vaccine and poor understanding of latency-associated determinants contribute to the failure of combating M.tb. The discovery of DosR as the master regulator of dormancy, opened new avenues to understand the pathophysiology of the bacterium. Though the specific functions of various DosR genes are yet to be discovered, they have been reported as potent T-cell activators and could elicit strong protective immune responses. Rv0569 is a DosR-encoded conserved hypothetical protein overexpressed during dormancy. However, it is not clearly understood how this protein modulates the host immune response. In the present study, we have demonstrated that Rv0569 has a high antigenic index and induces enhanced secretion of Th1 cytokines IL-12p40 and TNF-α as compared to Th2 cytokine IL-10 in macrophages. Mechanistically, Rv0569 induced the transcription of these pro-inflammatory signatures through the activation of NF-κB pathway. Further, immunization of mice with DosR protein Rv0569 switched the immune response towards Th1-biased cytokine pattern, characterized by the enhanced production of IFN-γ, IL-12p40, and TNF-α. Rv0569 augmented the expansion of antigen-specific IFN-γ and IL-2 producing effector CD4+and CD8+ T-cells which are hallmarks of Th1 biased protective immunity. Additionally, IgG2a/IgG1 and IgG2b/IgG1 ratio in the serum of immunized mice further confirmed the ability of Rv0569 to skew Th1 biased immune response. In conclusion, we emphasize that Rv0569 has the ability to generate signals to switch on Th1-dominated responses and further suggest that it could be a potential vaccine candidate against latent M.tb infection.

Neutrophil-Mediated Lung Injury Both via TLR2-Dependent Production of IL-1α and IL-12 p40, and TLR2-Independent CARDS Toxin after Mycoplasma pneumoniae Infection in Mice.

ABSTRACTMycoplasma pneumoniae (Mp) residing extracellularly in the respiratory tract is the primary cause of bacterial community-acquired pneumonia in humans. However, the detailed pathological mechanism of Mp infection, especially inflammation in the lung, remains unclear. This study examined the role of the neutrophils in the inflammation of Mp-induced pneumonia in mice and the mechanism of neutrophil infiltration into the lungs in the Mp-induced pneumonia. We observed massive infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) and lung injury after the Mp challenge. The neutrophils were shown to contribute to lung injury in Mp pneumonia but were not involved in eliminating Mp, suggesting that neutrophils are detrimental to the host in Mp pneumonia. Mp also induced the production of inflammatory cytokines and chemokines in the BALF in a toll-like receptor 2 (TLR2)-dependent manner. Particularly, both interleukin (IL)-1α and IL-12 p40 played a crucial role in neutrophil infiltration into the BALF in a coordinated manner. Both IL-1α and IL-12 p40 were released from the alveolar macrophages depending on the TLR2 and reactive oxygen species. In addition, the community-acquired respiratory distress syndrome (CARDS) toxin from Mp were found to induce neutrophil infiltration into BALF in a TLR2-independent and IL-1α-dependent manner. Collectively, the TLR2-dependent production of both IL-1α and IL-12 p40, and CARDS toxin have been elucidated to play an important role in neutrophil infiltration into the lungs subsequently leading to the lung injury upon Mp infection in mice. These data will aid in the development of therapeutics and vaccines for Mp pneumonia.IMPORTANCE Although Mp-induced pneumonia is usually a self-limiting disease, refractory life-threatening pneumonia is often induced. In addition, the development of alternative therapeutic strategies for Mp is expected because of the emergence of antibiotic-resistant Mp. However, the lack of knowledge regarding the pathogenesis of Mp-induced pneumonia, especially inflammation upon the Mp infection, makes it tedious to design novel therapeutics and vaccines. For example, although neutrophil infiltration is widely recognized as one of the characteristics of Mp-induced pneumonia, the precise role of neutrophils in the aggravation of Mp pneumonia remains unclear. This study showed that the infiltration of neutrophils in the lungs is detrimental to the host in Mp-induced pneumonia in mice. Furthermore, the TLR2-dependent IL-1α and IL-12 p40 production, and CARDS toxin play important roles in neutrophil infiltration into the lung, following lung injury. Our findings apply to the rational design of novel therapeutics and vaccines against Mp.

Human macrophage polarization shapes B. pertussis intracellular persistence.

We previously demonstrated that Bordetella pertussis, the etiologic agent of whooping cough, is able to survive inside human macrophages. The aim of this study was to examine the influence of macrophage polarization in the development of B. pertussis intracellular infections. To this end, primary human monocytes were differentiated into M1, M2a, or M2c macrophages and further infected with B. pertussis. Infected M1 macrophages showed a proinflammatory response evidenced by the production of TNF-α, IL-12p70, and IL-6. Conversely, infection of M2a and M2c macrophages did not induce TNF-α, IL-12p70, nor IL-6 at any time postinfection but showed a significant increase of M2 markers, such as CD206, CD163, and CD209. Interestingly, anti-inflammatory cytokines, like IL-10 and TGF-β, were induced after infection in the 3 macrophage phenotypes. B. pertussis phagocytosis by M1 macrophages was lower than by M2 phenotypes, which may be ascribed to differences in the expression level of B. pertussis docking molecules on the surface of the different phenotypes. Intracellular bactericidal activity was found to be significantly higher in M1 than in M2a or M2c cells, but live bacteria were still detected within the 3 phenotypes at the late time points after infection. In summary, this study shows that intracellular B. pertussis is able to survive regardless of the macrophage activation program, but its intracellular survival proved higher in M2 compared with the M1 macrophages, being M2c the best candidate to develop into a niche of persistence for B. pertussis.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1β, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

ERK5 modulates IL-6 secretion and contributes to tumor-induced immune suppression

Tumors exhibit a variety of strategies to dampen antitumor immune responses. With an aim to identify factors that are secreted from tumor cells, we performed an unbiased mass spectrometry-based secretome analysis in lung cancer cells. Interleukin-6 (IL-6) has been identified as a prominent factor secreted by tumor cells and cancer-associated fibroblasts isolated from cancer patients. Incubation of dendritic cell (DC) cultures with tumor cell supernatants inhibited the production of IL-12p70 in DCs but not the surface expression of other activation markers which is reversed by treatment with IL-6 antibody. Defects in IL-12p70 production in the DCs inhibited the differentiation of Th1 but not Th2 and Th17 cells from naïve CD4+ T cells. We also demonstrate that the classical mitogen-activated protein kinase, ERK5/MAPK7, is required for IL-6 production in tumor cells. Inhibition of ERK5 activity or depletion of ERK5 prevented IL-6 production in tumor cells, which could be exploited for enhancing antitumor immune responses.

The effects of dietary vitamin D supplementation and in vitro 1,25 dihydroxyvitamin D3 treatment on autophagy in bone marrow-derived dendritic cells from high-fat diet-induced obese mice

Obesity is associated with the dysregulation of vitamin D metabolism and altered immune responses in bone marrow-derived dendritic cells (BMDCs). Vitamin D can affect the differentiation, maturation, and activation of dendritic cells (DCs) and regulate autophagy via vitamin D receptor signaling. Autophagy was shown to be involved in the functions of DCs. We investigated the effects of dietary vitamin D supplementation and in vitro 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on autophagy in BMDCs from control diet (CON)-fed lean and high-fat diet (HFD)-induced obese mice. C57BL/6 male mice were fed CON or HFD with 10% or 45% kcal fat, respectively, supplemented with 1,000 or 10,000 IU vitamin D/kg diet (vDC or vDS) for 12 weeks. BMDCs were generated by culturing bone marrow cells from the mice with 20 ng/mL rmGM-CSF and treated with 1 nM 1,25(OH)2D3. Maturation of BMDCs was induced by lipopolysaccharide (50 ng/mL) stimulation. Treatment with 1,25(OH)2D3 inhibited the expression of phenotypes related to DC function (MHC class Ⅱ, CD86, CD80) and production of IL-12p70 by BMDCs from control and obese mice, regardless of dietary vitamin D supplementation. LC3Ⅱ/Ⅰ and VPS34 protein levels increased, and p62 expression decreased, after 1,25(OH)2D3 treatment of the BMDCs in CON-vDC only. Vdr mRNA levels decreased following 1,25(OH)2D3 treatment of BMDCs in the HFD-vDC. In conclusion, autophagy flux was increased by 1,25(OH)2D3 treatment of the BMDCs in CON-vDC but not in the HFD-vDC group. This suggests that the decreased expression of Vdr following 1,25(OH)2D3 treatment might have affected autophagy flux in BMDCs from obese mice.

Intranasal Administration of Codium fragile Polysaccharide Elicits Anti-Cancer Immunity against Lewis Lung Carcinoma.

Natural polysaccharides have shown promising effects on the regulation of immunity in animals. In this study, we examined the immune stimulatory effect of intranasally administered Codium fragile polysaccharides (CFPs) in mice. Intranasal administration of CFPs in C57BL/6 mice induced the upregulation of surface activation marker expression in macrophages and dendritic cells (DCs) in the mediastinal lymph node (mLN) and the production of interleukin-6 (IL-6), IL-12p70, and tumor necrosis factor-α in bronchoalveolar lavage fluid. Moreover, the number of conventional DCs (cDCs) was increased in the mLNs by the upregulation of C-C motif chemokine receptor 7 expression, and subsets of cDCs were also activated following the intranasal administration of CFP. In addition, the intranasal administration of CFPs promoted the activation of natural killer (NK) and T cells in the mLNs, which produce pro-inflammatory cytokines and cytotoxic mediators. Finally, daily administration of CFPs inhibited the infiltration of Lewis lung carcinoma cells into the lungs, and the preventive effect of CFPs on tumor growth required NK and CD8 T cells. Furthermore, CFPs combined with anti-programmed cell death-ligand 1 (PD-L1) antibody (Ab) improved the therapeutic effect of anti-PD-L1 Ab against lung cancer. Therefore, these data demonstrated that the intranasal administration of CFP induced mucosal immunity against lung cancer.

Host defense against Neospora caninum infection via IL-12p40 production through TLR2/TLR3-AKT-ERK signaling pathway in C57BL/6 mice

Neospora caninum is an intracellular parasite which can cause neosporosis and significant economic losses in both dairy and beef industries worldwide. A better understanding of the immune response by host cells against N. caninum could help to design better strategies for the prevention and treatment of neosporosis. Although previous studies have shown TLR2/TLR3 were involved in controlling N. caninum infection in mice, the precise mechanisms of the AKT and MAPK pathways controlled by TLR2/TLR3 to regulate N. caninum-induced IL-12p40 production and the role of TLR2/TLR3 in anti-N. caninum infection in bovine macrophages remain unclear. In the present study, TLR2−/− mice displayed more parasite burden and lower level of IL-12p40 production compared to TLR3−/− mice. N. caninum could activate AKT and ERK signaling pathways in WT mouse macrophages, which were inhibited in TLR2−/− and TLR3−/− mouse macrophages. In N. caninum-infected WT mouse macrophages, AKT inhibitor or AKT siRNA could decrease the phosphorylation of ERK. AKT or ERK inhibitors reduced the production of IL-12p40 and increased the number of parasites. The productions of ROS, NO, and GBP2 were significantly reduced in TLR2−/− and TLR3−/− mouse macrophages. Supplementation of rIL-12p40 inhibited N. caninum proliferation and rescued the productions of IFN-γ, NO, and GBP2 in WT, TLR2−/−, and TLR3−/− mouse macrophages. In bovine macrophages, the expressions of TLR2, TLR3, and IL-12p40 mRNA were significantly enhanced by N. caninum, and N. caninum proliferation was inhibited by TLR2/TLR3 agonists. Taken together, the proliferation of N. caninum in mouse macrophages was controlled by the TLR2/TLR3-AKT-ERK signal pathway via increased IL-12p40 production, which in turn lead to the productions of NO, GBP2, and IFN-γ during N. caninum infection. And in bovine macrophages, TLR2 and TLR3 contributed to inhibiting N. caninum proliferation via increased IL-12p40 production.

Tumour-Derived Reg3A Educates Dendritic Cells to Promote Pancreatic CancerProgression.

As a pancreatic inflammatory marker, regenerating islet-derived protein 3A (Reg3A) plays a key role in inflammation-associated pancreatic carcinogenesis by promoting cell proliferation, inhibiting apoptosis, and regulating cancer cell migration and invasion. This study aimed to reveal a novel immuno-regulatory mechanism by which Reg3A modulates tumour-promoting responses during pancreatic cancer (PC) progression. In an in vitro Transwell system that allowed the direct co-culture of human peripheral blood-derived dendritic cells (DCs) and Reg3A-overexpressing/ silenced human PC cells, PC cell-derived Reg3A was found to downregulate CD80, CD83 and CD86 expression on educated DCs, increase DC endocytic function, inhibit DC-induced T lymphocyte proliferation, reduce IL-12p70 production, and enhance IL-23 production by DCs. The positive effect of tumour-derived Reg3A-educated human DCs on PC progression was demonstrated in vivo by intraperitoneally transferring them into PC-implanted severe combined immunodeficiency (SCID) mice reconstituted with human T cells. A Reg3A-JAK2/STAT3 positive feedback loop was identified in DCs educated with Reg3A. In conclusion, as a tumour-derived factor, Reg3A acted to block the differentiation and maturation of the most important antigen-presenting cells, DCs, causing them to limit their potential anti-tumour responses, thus facilitating PC escape and progression.

Early Immune Initiation by Porcine Cells following Toxoplasma gondii Infection versus TLR Ligation.

Containment of acute Toxoplasma gondii infection is dependent on an efficient interferon gamma response. However, the earliest steps of immune response initiation immediately following exposure to the parasite have not been previously characterized in pigs. Murine and human myeloid cells produce large quantities of interleukin (IL)-12 during early T. gondii infection. We therefore examined IL-12 expression by porcine peripheral blood monocytes and dendritic cell (DC) subsets following toll-like receptor (TLR) ligation and controlled T. gondii tachyzoite infection. We detected IL-12p40 expression by porcine plasmacytoid DC, but not conventional or monocyte-derived DC following TLR ligation. Unexpectedly, we also observed considerable IL-12p40 production by porcine CD3– NKp46+ cells—a classical natural killer cell phenotype—following TLR ligation. However, in response to T. gondii exposure, no IL-12 production was observed by either DC or CD3– NKp46+ cells. Despite this, IL-18 production by DC-enriched peripheral blood mononuclear cells was detected following live T. gondii tachyzoite exposure. Only combined stimulation of porcine peripheral blood mononuclear cells with recombinant IL-12p70 and IL-18 induced innate interferon gamma production by natural killer cells, while T cells and myeloid cells did not respond. Therefore, porcine CD3– NKp46+ cells serve as important IL-12 producers following TLR ligation, while IL-18 likely plays a prominent role in early immune response initiation in the pig following T. gondii infection.

CGAS-STING and MyD88 Pathways Synergize in Ly6Chi Monocyte to Promote Streptococcus pneumoniae-Induced Late-Stage Lung IFNγ Production.

The cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS–STING) pathway senses DNA and induces type I interferon (IFN) production. Whether and how the STING pathway crosstalk to other innate immune pathways during pathogen infection, however, remains unclear. Here, we showed that STING was needed for Streptococcus pneumoniae-induced late, not early, stage of lung IFNγ production. Using knockout mice, IFNγ reporter mice, intracellular cytokine staining, and adoptive cell transfer, we showed that cGAS–STING-dependent lung IFNγ production was independent of type I IFNs. Furthermore, STING expression in monocyte/monocyte-derived cells governed IFNγ production in the lung via the production of IL-12p70. Surprisingly, DNA stimulation alone could not induce IL-12p70 or IFNγ in Ly6Chi monocyte. The production of IFNγ required the activation by both DNA and heat-killed S. pneumococcus. Accordingly, MyD88−/− monocyte did not generate IL-12p70 or IFNγ. In summary, the cGAS–STING pathway synergizes with the MyD88 pathway in monocyte to promote late-stage lung IFNγ production during pulmonary pneumococcal infection.

Phenolic Constituents from Platycodon grandiflorum Root and Their Anti-Inflammatory Activity.

Six lignols (1–6), including two new compounds (+)-(7R,8R)-palmitoyl alatusol D (1) and (+)-(7R,8R)-linoleyl alatusol D (2), along with four phenolics (7–10), a neolignan (11), three alkyl aryl ether-type lignans (12–14), two furofuran-type lignans (15–16), three benzofuran-type lignans (17–19), a tetrahydrofuran-type lignan (20), and a dibenzylbutane-type lignan (21) were isolated from the ethyl acetate-soluble fraction of the methanol extract of Platycodon grandiflorum (Jacq.) A. DC. root. The chemical structures of the obtained compounds were elucidated via high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy analyses. The obtained spectroscopic data agreed well with literature. Among the isolated compounds, eighteen (1–7 and 11–21) were isolated from P. grandiflorum and the Campanulaceae family for the first time. This is the first report on lignol and lignan components of P. grandiflorum. The anti-inflammatory effects of the isolated compounds were examined in terms of their ability to inhibit the production of pro-inflammatory cytokines IL-6, IL-12 p40, and TNF-α in lipopolysaccharide-stimulated murine RAW264.7 macrophage cells. Nine compounds (4–6, 12, and 15–19) exhibited inhibitory effects on IL-12 p40 production, eleven compounds (1–6, 12, 15–17, and 19) exhibited inhibitory activity on IL-6 production, and eleven compounds (1–6 and 15–19) exhibited inhibitory effects against TNF-α. These results warrant further investigation into the potential anti-inflammatory activity and general benefits of the phenolic constituents of P. grandiflorum root.

In vitro interaction between murine carcinoma cells and macrophages after treatment with Carbo animalis

Clinical reports show improvement of the quality of life of cancer patients following treatment with homeopathic medicine Carbo animalis, although the literature on this subject is still rare and the mechanisms of action unknown. Previous results obtained by our group indicated that treatment of mice with Carbo animalis 6cH increased the migration of macrophages and B1 cells (phagocyte progenitors) to Ehrlich tumor primary site. In the present study we analyzed the interaction of macrophages with murine breast cancer cells (4T1) after treatment with Carbo animalis in different homeopathic dilutions: 6cH, 30cH and combination 30cH + 6cH (MIX). All dilutions and vehicles were physically and chemically analyzed for conductance and composition of microparticles in suspension by means of energy-dispersive x-ray spectroscopy (EDX). Exposure of macrophages co-cultured with 4T1 cells to treatments over a period of 48 hours led to the following significant changes: 1) reduction of TNF-alpha production in co-cultures compared to simple macrophage culture, indicating an inhibitory effect of the tumor on macrophage activity; b) higher expression of pseudopodia and IL-6 production after exposure to all treatments; c) increase of the cell number per microscopic field, increase in the interaction (cell-cell contact) between macrophages and 4T1 cells and increase in production of GM-CSF, IL-12p40 and MCP-1 after treatment with Carbo animalis 6cH; d) increase of VEGF production in cells treated with Carbo animalis 30cH. There was no change in nitric oxide production with any treatment. Physical-chemical analysis of the drugs revealed significant increase in the conductivity of Carbo animalis 30cH and MIX samples compared to the vehicle (30% alcohol). The microparticles exhibited an extremely diverse composition, including Na, Si, Cl, F, Pb, S, Ca, Mg, Al, K, Nb, B and Be . Only Carbo animalis 6cH exhibited boron and beryllium with frequency above 50% per particle, and this drug induced the most significant changes on the macrophage-tumor interaction. However, the role of these elements in the immunomodulatory activity observed both in vivo and in vitro still deserves further investigation.

AeMOPE-1, a Novel Salivary Peptide From Aedes aegypti, Selectively Modulates Activation of Murine Macrophages and Ameliorates Experimental Colitis.

The sialotranscriptomes of Aedes aegypti revealed a transcript overexpressed in female salivary glands that codes a mature 7.8 kDa peptide. The peptide, specific to the Aedes genus, has a unique sequence, presents a putative secretory nature and its function is unknown. Here, we confirmed that the peptide is highly expressed in the salivary glands of female mosquitoes when compared to the salivary glands of males, and its secretion in mosquito saliva is able to sensitize the vertebrate host by inducing the production of specific antibodies. The synthetic version of the peptide downmodulated nitric oxide production by activated peritoneal murine macrophages. The fractionation of a Ae. aegypti salivary preparation revealed that the fractions containing the naturally secreted peptide reproduced the nitric oxide downmodulation. The synthetic peptide also selectively interfered with cytokine production by murine macrophages, inhibiting the production of IL-6, IL-12p40 and CCL2 without affecting TNF-α or IL-10 production. Likewise, intracellular proteins associated with macrophage activation were also distinctively modulated: while iNOS and NF-κB p65 expression were diminished, IκBα and p38 MAPK expression did not change in the presence of the peptide. The anti-inflammatory properties of the synthetic peptide were tested in vivo on a dextran sulfate sodium-induced colitis model. The therapeutic administration of the Ae. aegypti peptide reduced the leukocytosis, macrophage activity and nitric oxide levels in the gut, as well as the expression of cytokines associated with the disease, resulting in amelioration of its clinical signs. Given its biological properties in vitro and in vivo, the molecule was termed Ae des-specific MOdulatory PEptide (AeMOPE-1). Thus, AeMOPE-1 is a novel mosquito-derived immunobiologic with potential to treat immune-mediated disorders.

Decreased Production of TNF-α and IL-6 Inflammatory Cytokines in Non-Pregnant Idiopathic RPL Women Immunomodulatory Effect of Sildenafil Citrate on the Cellular Response of Idiopathic RPL Women.

Sildenafil citrate (SC), a PDE5 inhibitor, a drug for erectile dysfunction (ED) and pulmonary hypertension (PAH), was found to exert a positive effect on pregnancy outcomes when administered intravaginally before conception. In our previous studies, sildenafil increased endometrial thickness and significantly decreased peripheral blood NK cell activity after the intravaginal administration in women with recurrent pregnancy loss (RPL). No data are available to confirm the effect of sildenafil on maternal T cell populations involved in shaping fetal-maternal tolerance and NK cell activity. Thus, the present study aimed to establish if SC influences NKT cells or the axis of Th17/Treg cells and Th1/Th2 cytokine production. Materials and methods: Twenty-one healthy fertile women and twenty-two nonpregnant women with idiopathic RPL were studied. The ELISA method was used to evaluate the production of cytokines, including IL-2, IL-12p40, IL-4, IL-10, IL-6, IL-17, IL-21, TGF-β, TNF-α, and IFN-γ in PBMC culture supernatants before and after supplementation with the physiological concentration of SC. The percentages of NKT (CD56+CD3+CD44+CD161+), Treg (CD4+CD25+FOXP3+) and Th17 (CD4+CD25+IL-17A+) cells were determined with flow cytometry method. Results: Unexpectedly, we found that the PBMCs of patients with RPL produced a significantly lower level of inflammatory cytokines (TNF-α and IL-6) and a higher level of anti-inflammatory cytokines (TGF-β and IL-10). SC significantly decreased IL-6, IL-12 and increased TGF-β cytokine concentration in fertile women. In the case of RPL patients’ PBMCs, SC improved the production of TNF-α and IL-10. Conclusions: Lower concentration of proinflammatory cytokines in idiopathic RPL women compared to fertile women might suggest the exhaustion of the immune system. The emphasized production of IL-10 by SC partially explains the previously observed downregulation of NK cell activity in RPL patients. The immunomodulatory effect of the drug might be utilized in anti-inflammatory therapies and help achieve positive pregnancy outcomes in women with reproductive failure due to a Th1/Th2 imbalance.