Cultures Of Peritoneal Macrophages Research Articles

CYTOKINE-MODULATING INFLUENCE OF HIPEC ON THE INTRAPERITONEAL HOMEOSTASIS FORMATION IN PATIENTS WITH OVARIAN CANCER

Background: Ovarian cancer (OC) has the greatest mortality rate among oncogynecological diseases. Most cases are diagnosed at the peritoneal dissemination stage, resulting in radical treatment. Most cases (75%) are diagnosed in III-IV stages at the stage of peritoneal dissemination, making the radical treatment impossible. Intraoperative hyperthermic intraperitoneal chemoperfusion (HIPEC), which has ceased to be an experimental technique over the past decade, is increasingly used among the methods of combating peritoneal carcinomatosis and demonstrates an increase in recurrence-free and overall survival in advanced stages of OC. Nevertheless, certain pathophysiological aspects of the effect of HIPEC on intra-abdominal homeostasis, and therefore on the further course of the disease, have not been elucidated. However, understanding this effect may be the key to the successful application of HIPEC and predicting its efficacy in each case of OC.
 Objectives: To access changes in intraperitoneal homeostasis in patients with advanced OC after HIPEC procedure (as a stage of cytoreductive surgery); in particular, to evaluate changes of TNF and TGF-β expression under the influence of HIPEC and systemic chemotherapy in the OC microenvironment.
 Materials and methods: The study included 33 OC patients treated at Lviv Regional Cancer Centre in 2016–2020. Twelve of them received HIPEC. The primary tumor cultures and primary cultures of peritoneal mesothelium and macrophages were obtained for all patients. The cytokines TNF and TGF-β activity were determined in their conditional media.
 Results: The decrease of TNF and TGF-β concentrations in the drainage exudate after HIPEC was revealed compared with the same parameters in the ascitic fluid before the operation. One-hour hyperthermia of the primary culture of peritoneal ascites-associated macrophages of recurrent OC patients decreased the TNF level in conditional media. A decline was found in 10 out of 12 cases.
 Conclusions: HIPEC has a cytokine-modulating effect on the intra-abdominal homeostasis of patients with OC, suppressing TNF expression by peritoneal macrophages. Reduced activity of TNF in supernatants of OC primary tumor cultures was associated with higher sensitivity to chemotherapy.

Necrotic cardiac myocytes skew macrophage polarization towards a classically activated phenotype.

Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1β secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1β. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

Is the production of reactive oxygen and nitrogen species by macrophages associated with better infectious control in female mice with experimentally disseminated and pulmonary mucormycosis?

Different levels of resistance against Rhizopus oryzae infection have been observed between inbred (BALB/c) and outbred (Swiss) mice and are associated with the genetic background of each mouse strain. Considering that macrophages play an important role in host resistance to Rhizopus species, we used different infectious outcomes observed in experimental mucormycosis to identify the most efficient macrophage response pattern against R. oryzae in vitro and in vivo. For this, we compared BALB/c and Swiss macrophage activity before and after intravenous or intratracheal R. oryzae infections. The production of hydrogen peroxide (H2O2) and nitric oxide (NO) was determined in cultures of peritoneal (PMΦ) or alveolar macrophages (AMΦ) challenged with heat-killed spores of R. oryzae. The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) were measured to confirm our findings. Naïve PMΦ from female BALB/c mice showed increased production of H2O2, TNF-α, and IL-10 in the presence of heat-killed spores of R. oryzae. Naïve PMΦ from female Swiss mice were less responsive. Naïve AMΦ from the two strains of female mice were less reactive to heat-killed spores of R. oryzae than PMΦ. After 30 days of R. oryzae intravenous infection, lower fungal load in spleen from BALB/c mice was accompanied by higher production of H2O2 by PMΦ compared with Swiss mice. In contrast, AMΦ from BALB/c mice showed higher production of NO, TNF-α, and IL-10 after 7 days of intratracheal infection. The collective findings reveal that, independent of the female mouse strain, PMΦ is more reactive against R. oryzae upon first contact than AMΦ. In addition, increased PMΦ production of H2O2 at the end of disseminated infection is accompanied by better fungal clearance in resistant (BALB/c) mice. Our findings further the understanding of the parasite-host relationship in mucormycosis.

The effect of endomorphins-1, -2 on the production of reactive oxygen species and the absorption activity of innate immunity cells <i>in vitro</i> and <i>in vivo</i>

Background. A high percentage of people in the world suffer from acute or chronic pain, the treatment of which is based on the introduction of morphine and its derivatives, agonists of the µ-opioid receptors, leading to the development of addiction in patients. An urgent task is to study the properties of endogenous opioid peptides, which are capable of showing not only pronounced analgesic activity, but also have a number of other effects on the periphery, while having a much smaller spectrum of side effects. Of particular interest are the immunomodulatory effects of peptide agonists of µ-receptors – endomorphins, tetrapeptides, the effects of which are not described in detail in the literature. Due to their structure and properties, endorphins can be considered as potential substitutes for low-molecular-weight opiates. The aim. To investigate the effect of endorphins-1,2 on the synthesis of oxygen radicals and the absorption activity of innate immunity cells in vitro, in vivo. Materials and methods. The object of the in vitro study is peripheral venous blood leukocytes from donors aged 22–40 years; the object of the in vivo study is peritoneal flush cells of Swiss white male mice. Oxygen-dependent microbicidal activity was evaluated using a luminol-dependent chemiluminescence reaction. Phagocytic activity was evaluated by flow cytometry. Results. It has been found that endomorphins inhibited spontaneous production of reactive oxygen species in vitro, in vivo. Endomorphin-2 reduced the intensity of respiratory explosion in stimulated cultures of peritoneal macrophages. Blockade of opiate receptors with naloxone in vitro, in vivo did not cancel the effects of endomorphins. Both peptides and naloxone had no effect on the phagocytic activity of macrophages of the peritoneal cavity of mice in vivo, but in vitro endomorphins led to an increase in the percentage of phagocytosis of peripheral blood leukocytes. Conclusions. The effects of endomorphins in vitro, in vivo are predominantly depressive.

Molecular Hybridization Strategy on the Design, Synthesis, and Structural Characterization of Ferrocene-N-acyl Hydrazones as Immunomodulatory Agents.

Immunomodulatory agents are widely used for the treatment of immune-mediated diseases, but the range of side effects of the available drugs makes necessary the search for new immunomodulatory drugs. Here, we investigated the immunomodulatory activity of new ferrocenyl-N-acyl hydrazones derivatives (SintMed(141−156). The evaluated N-acyl hydrazones did not show cytotoxicity at the tested concentrations, presenting CC50 values greater than 50 µM. In addition, all ferrocenyl-N-acyl hydrazones modulated nitrite production in immortalized macrophages, showing inhibition values between 14.4% and 74.2%. By presenting a better activity profile, the ferrocenyl-N-acyl hydrazones SintMed149 and SintMed150 also had their cytotoxicity and anti-inflammatory effect evaluated in cultures of peritoneal macrophages. The molecules were not cytotoxic at any of the concentrations tested in peritoneal macrophages and were able to significantly reduce (p < 0.05) the production of nitrite, TNF-α, and IL-1β. Interestingly, both molecules significantly reduced the production of IL-2 and IFN-γ in cultured splenocytes activated with concanavalin A. Moreover, SintMed150 did not show signs of acute toxicity in animals treated with 50 or 100 mg/kg. Finally, we observed that ferrocenyl-N-acyl hydrazone SintMed150 at 100 mg/kg reduced the migration of neutrophils (44.6%) in an acute peritonitis model and increased animal survival by 20% in an LPS-induced endotoxic shock model. These findings suggest that such compounds have therapeutic potential to be used to treat diseases of inflammatory origin.

The effect of enzymatic hydrolysate of lymphoid tissue of broiler chickens on the morphofunctional state of macrophages

The goal of studying the mechanisms of action of cells that structure the immune system is to obtain additional data about the influence of immunomodulating peptides of natural origin on the possibility of regulating immune processes. The effect of enzymatic hydrolysate of lymphoid tissue of broiler chickens (EHLTBC) on the viability of culture of peritoneal and alveolar macrophages, on the morphological characteristics and functional activity of macrophages has been studied. The effect of EHLTBC on the viability of culture of peritoneal and alveolar macrophages has been investigated. Morphometric characteristics of macrophages under the influence of EHLTBC have been given. The functional activity of peritoneal and alveolar macrophages has been assessed against the background of the introduction of EHLTBC. Enzymatic hydrolysate of lymphoid tissue of broiler chickens at concentrations of 1, 5 and 10 % does not have a toxic effect on the culture of alveolar and peritoneal macrophages. EHLTBC enhances the phagocytic activity of peritoneal macrophages, which enhances their role in the chemotaxis and immune response. The stimulating effect of EHLTBC of poultry on the ability of macrophages to adhere has been revealed.

Nanoparticle Internalization Promotes the Survival of Primary Macrophages.

Macrophages, a class of tissue resident innate immune cells, are responsible for sequestering foreign objects through the process of phagocytosis, making them a promising target for immune-modulation via particulate engineering. Here, we report that nanoparticle (NP) dosing and cellular internalization via phagocytosis significantly enhances survival of ex vivo cultures of primary bone marrow-derived, alveolar, and peritoneal macrophages over particle-free controls. The enhanced survival is attributed to suppression of caspase-dependent apoptosis and is linked to phagocytosis and lysosomal signaling. Uniquely, poly(ethylene glycol)-based NP treatment extended cell viability in the absence of macrophage polarization and enhanced expression of pro-survival B cell lymphoma-2 (Bcl-2) protein in macrophages following multiple routes of in vivo administration. The enhanced survival phenomenon is also applicable to NPs of alternative chemistries, indicating the potential universality of this phenomenon with relevant drug delivery particles. These findings provide a framework for extending the lifespan of primary macrophages ex vivo for drug screening, vaccine studies, and cell therapies and has implications for any in vivo particulate immune-engineering applications.

The effect of coal-derived humic substances and their silver-containing bionanocomposites on arginine balance in peritoneal macrophages of intact mice

Background. Antigen-presenting cells (APCs), especially macrophages, play an important role in the body defense against various pathogens. Their dysfunction and polarization are associated with most inflammatory and autoimmune diseases. The inflammatory process is regulated by activation and / or inhibition of genes differentially expressed by macrophages. Successful correction of inflammation leads firstly to elimination of inflammatory stimuli and then to remodeling and restoration of tissues and organs. It was experimentally confirmed that silvercontaining bionanocomposites based on natural humic substances (HS) obtained from coal of different origin, as well as initial matrices of these HS, are capable of activating pro- and anti-inflammatory properties of macrophages.Aim. To study cytotoxic, pyrogenic, and immunomodulatory properties (arginine balance) of initial HS samples and samples of silver nanoparticles ultradispersed in these HS matrices (HS-AgNPs) in the cell culture of peritoneal macrophages, as well as their effect on pro- and anti-inflammatory properties of APCs.Materials and methods. Cultural and biochemical methods were used in the study.Results. The study showed that the samples CHE-K, CHE-AgNPs, CHS-K, and CHP-K increased M1 macrophage polarization due to stimulation of the NO-synthase activity and inhibition of arginase. The samples CHI-K, CHIAgNPs, CHP-AgNPs, and CHS-AgNPs modulated an alternative M2 or M2-like state of macrophage activation. At the same time, HS are not cytotoxic at effective concentrations, and three out of four studied samples did not contain pyrogenic impurities.Conclusion. The use of HS and their silver-containing bionanocomposites, which have the ability to greatly affect the polarization of antigen-presenting cells, is a promising research area in correction of the inflammatory response for solving an important social and medical problem of treating chronic wounds.

The Influence of Water-Soluble Polysaccharides of Crataegus sanguinea Pall. on Nitric Oxide Production by Macrophages.

The in vitro addition of water-soluble polysaccharides isolated from the leaves of Crataegus sanguinea Pall. to culture of mouse peritoneal macrophages induced classical activation of antigen-presenting cells by increasing NO synthase activity and reducing arginase expression.

In Vitro Study of the Expression of CD1, CD14, CD25, CD30, CD35, CD95 Receptors by Macrophages of Mice Infected with Mycobacterium tuberculosis.

In cultures of peritoneal macrophages (MP) of male BALB/c mice infected with Mycobacterium tuberculosis from the BCG vaccine, the expression of CD1, CD14, CD25, CD30, CD35, and CD95 receptors was studied in vitro 3 months after infection. In MP cultures from intact and infected mice, mononuclear MP predominated (96 and 92%, respectively). Bi- and trinuclear MP in MP cultures from control and infected mice constituted 4 and 8.3% of all MP, respectively. In the cultures of both groups, no obvious correlations between the number of MP expressing CD-receptors and number of nuclei in these cells were found, but the expression of CD14 receptor was more often noted. In cultures from infected animals, hypertrophied MP and enhanced (by several times) expression of all CD-receptors were observed. The increase in the expression of CD-receptor can be determined by activation of plastic processes in hypertrophied MP (in epithelioid and in numerically insignificant polynuclear MP), which is due to the phenomenon of prolonged M. tuberculosis persistence in the vacuolar apparatus of these cells.

Medicinal Plants from Brazilian Cerrado Biome: Potential sources of new anti-inflammatory compounds and antitumor agents on Ehrlich carcinoma.

This work describes a pharmacological screening of Brazilian medicinal plants through their anti-inflammatory and cytotoxicity activities. Cytotoxicity activity of Mouriri elliptica and Alchornea glandulosa as well as the drugs celecoxib and doxorubicin were evaluated in cultures of peritoneal macrophages. The immune system influence of these samples was analyzed by determining production/inhibition of NO, production of tumor necrosis factor-α and production of interleukin-10. Regarding the production/inhibition of NO, there was NO production by M. elliptica and NO inhibition when the cells were exposed to A. glandulosa; Macrophages generally produce more NO, plus TNF-α and less IL-10, when associated to the tumor phenomenon, characterizing the inflammation involved in cancer. A. glandulosa showed anti-inflammatory effect, inhibited NO production and it was associated with low TNF-α production, although not as low as the macrophages associated with celecoxib and doxorubicin. These cytokines were not different in animals with tumor. Celecoxib confirms its anti-inflammatory action by markedly inhibiting NO and TNF-α, but also inhibiting IL-10 which is an anti-inflammatory cytokine. Doxorubicin inhibited NO in a higher percentage in the group of animals with tumor, although the literature reports that this drug stimulates the production of NO and this collaborates with its cytotoxic effect.

The administration of low-dose Curcuma longa extract induces M2 polarization in peritoneal macrophage culture

Curcuma longa (turmeric) has been widely used to accelerate wound healing, but the underlying mechanism remains unclear. Wound healing consists of four phases which are correlated and overlapping, i.e., coagulation, inflammation, proliferation and remodeling. Macrophages play an important role in most phases. Macrophage polarization to M2 initiates the proliferation phase, which is characterized by the production of several crucial growth factors. Since turmeric has been known to be an herb that accelerates wound closure, we investigated whether Curcuma longa extract administration in macrophage culture induces M2 macrophage switching. An in vitro study was performed using peritoneal macrophages from Swiss Webster strain mice. Peritoneal cells were collected and cultured in a 24-well plate. After 2 hours of incubation, macrophages (adherent cells) were treated with 0.5 ppm, 1.0 ppm and 5.0 ppm of ethanolic extract of Curcuma longa and incubated for 2 days. Quantitative real time PCR was performed to quantify M2 and M1 marker gene expression. The results revealed the upregulation of M2 marker (Arginase-1) expression upon administration of 0.5 ppm of Curcuma longa extract, but not of higher doses (1.0 and 5.0 ppm). In parallel, the ratio of Arg-1/Inos was high upon administration of 0. 5ppm of extract. In conclusion, Curcuma longa extract induces in vitro M2 polarization in low-dose administration.

Влияние водорастворимых полисахаридов Crataegus sanguinea Pall. на продукцию оксида азота макрофагами

The in vitro addition of water-soluble polysaccharides isolated from the leaves of Crataegus sanguinea Pall. to culture of mouse peritoneal macrophages induced classical activation of antigen-presenting cells by increasing NO synthase activity and reducing arginase expression.

Study of Immunotropic Properties of Water-Soluble Polysaccharides Isolated from Conium maculatum Grass.

Addition of water-soluble polysaccharides isolated from Conium maculatum L. to the mouse peritoneal macrophage culture induces classical activation of antigen-presenting cells due to an increase in NO synthase activity and a decrease in arginase expression.

Molecular basis of carrageenan-induced cytokines production in macrophages

BackgroundLow molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production.MethodsPrimary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed.ResultsHere we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1β. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1β relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1β into IL-1β is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components.ConclusionsIn conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies.1e-L4pWBc1sacKrQDF6Cf5Video abstractGraphical Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1β. The Cg-stimulated macrophages produces pro-IL-1β depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1β is dependent on the canonical NLRP3 inflammasome.

Clomipramine, a tricyclic antidepressant, and selegiline, a monoamine oxidase-B inhibitor, modulate the activity of phagocytic cells after oral administration in mice.

Our aim was to find out whether clomipramine, a tricyclic antidepressant, and selegiline, a monoamine oxidase-B inhibitor, influence the activity of phagocytic cells after in-vivo administration in mice. Clomipramine and selegiline were administered to Balb/c mice orally at a dose of 1mg/kg, 7 or 14 times. IL-1β and nitric oxide (NO) levels were measured in supernatants of the peritoneal macrophage cultures stimulated in vitro with lipopolysaccharide from Escherichia coli. The phagocytic activity of the granulocytes and monocytes was determined using a commercial Phagotest 24 and 72h after the last dose of the investigated drugs. Seven doses of clomipramine or selegiline decreased IL-1β production, while a rise in its synthesis was observed after 14 doses of selegiline. Clomipramine administered 14 times increased NO production. Clomipramine and selegiline administered seven times reduced the percentage of phagocytosing granulocytes. The drugs administered 14 times increased the percentage of phagocytosing granulocytes and decreased the percentage of phagocytosing monocytes. Both clomipramine and selegiline administered in vivo changed the phagocytic activity of blood cells and IL-1β and NO production by murine peritoneal macrophages. This effect depended on the drug, the number of doses and the type of phagocytic cells.

Diesel exhaust particles dysregulate multiple immunological pathways in murine macrophages: Lessons from microarray and scRNA-seq technologies

Exposure to ambient particulate matter has been shown to promote a variety of disorders, including cardiovascular diseases predominantly of ischemic etiology. However, the mechanisms linking inhaled particulates with systemic vascular effects, resulting in worsened atherosclerosis, are not well defined. We assessed the potential role of macrophages in translating these effects by analyzing gene expression patterns in response to diesel exhaust particles (DEP) at the average cell level, using Affymetrix microarrays in peritoneal macrophages in culture (in vitro), and at the individual cell level, using single-cell RNA sequencing (scRNA-seq) in alveolar macrophages collected from exposed mice (in vivo). Peritoneal macrophages were harvested from C57BL/6J mice and treated with 25 μg/mL of a DEP methanol extract (DEPe). These cells exhibited significant (FDR < 0.05) differential expression of a large number of genes and enrichment in pathways, especially engaged in immune responses and antioxidant defense. DEPe led to marked upregulation of heme oxygenase 1 (Hmox1), the most significantly upregulated gene (FDR = 1.75E-06), and several other antioxidant genes. For the in vivo work, C57BL/6J mice were subjected to oropharyngeal aspiration of 200 μg of whole DEP. The gene expression profiles of the alveolar macrophages harvested from these mice were analyzed at the single-cell level using scRNA-seq, which showed significant dysregulation of a broad number of genes enriched in immune system pathways as well, but with a large heterogeneity in how individual alveolar macrophages responded to DEP exposures. Altogether, DEP pollutants dysregulated immunological pathways in macrophages that may mediate the development of pulmonary and systemic vascular effects.

KLF4 upregulation is involved in alternative macrophage activation during secondary Echinococcus granulosus infection.

The objective of this study was to investigate macrophage polarization during the early stages of secondary Echinococcus granulosus sensu lato (E.granulosus s.l.) infection. We observed an early initial increase in inflammatory genes (peaking at 5-10days) and a later rise in M (IL-4)-like genes (still rising by day 15). In addition, we showed that the induction of M (IL-4)-like genes was paralleled by an increase in expression of the transcription factor KLF4. Most of the changes observed in vivo were reproduced in vitro upon the culture of normal peritoneal macrophages with live E.granulosus s.l. protoscoleces (PSC), and that knockdown of KLF4 in this system attenuates M (IL-4) differentiation. Our results suggest that KLF4 pathway contributes to the differentiation of macrophages towards M (IL-4)-like phenotype during early stages of secondary E.granulosus s.l. infection.

Immunomodulatory role of nonneuronal cholinergic signaling in myocardial injury

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Recombinant mouse calcitonin gene-related peptide secreted by Lactococcus lactis inhibits lipopolysaccharide-induced inflammatory response in macrophages.

We describe the development of a genetically modified strain of lactic acid bacteria (gmLAB) capable of producing a recombinant mouse calcitonin gene-related peptide (rCGRP). This strain (NZ-CGRP) was generated by introducing a CGRP secretion plasmid into Lactococcus lactis NZ9000. Western blotting confirmed the secretion of rCGRP in the presence of the inducer nisin. Highly purified rCGRP was obtained from the culture supernatants of NZ-CGRP. We demonstrated that prophylactic exposure of a culture of mouse peritoneal macrophages to rCGRP inhibited lipopolysaccharide (LPS) induction of tumor necrosis factor-α (TNF-α). The rCGRP-secreting gmLAB strain holds promise for development as a new anti-inflammatory prophylactic.