In Vitro and In Vivo Evaluation of Virus-Induced Innate Immunity in Mouse.

The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-ß) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo.

A new model and precious tool to study molecular mechanisms of macrophage aging.

The accumulation of senescent cells, characterized by a senescence-associated secretory phenotype (SASP), contributes to chronic inflammation and age-related diseases (ARD). During aging, macrophages can adopt a senescent-like phenotype and an altered function, which promotes senescent cell accumulation. In the context of aging and ARD, controlling the resolution of the inflammatory response and preventing chronic inflammation, especially by targeting macrophages, must be a priority. Aging being a dynamic process, we developed a model of in vitro murine peritoneal macrophage aging. Our results show that macrophages cultured for 7 or 14 days exhibit a senescence-like phenotype: proliferation decrease, the levels of cyclin-dependent kinase inhibitors p16INK4A and p21CIP1 and of pro-inflammatory SASP components (MCP-1, IL-6, IL-1ß, TNF-α, and MMP-9) increase, phagocytosis capacity decline and glycolytic activity is induced. In our model, chronic treatment with CB3, a thioredoxin-1 mimetic anti-inflammatory peptide, completely prevents p21CIP1 increase and enables day 14 macrophages to maintain proliferative activity.We describe a new model of macrophage aging with a senescence-like phenotype associated with inflammatory, metabolic and functional perturbations. This model is a valuable tool for characterizing macrophage aging mechanisms and developing innovative strategies with promising therapeutical purpose in limiting inflammaging and ARD.

SPA Promotes Atherosclerosis Through Mediating Macrophage Foam Cell Formation-Brief Report.

BACKGROUND: Atherosclerosis is a progressive inflammatory disease in which macrophage foam cells play a central role in disease pathogenesis. SPA (surfactant protein A) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated. METHODS: SPA expression was assessed in healthy and atherosclerotic human coronary arteries and the brachiocephalic arteries of wild-type or ApoE-deficient mice fed high-fat diets for 4 weeks. Hypercholesteremic wild-type and SPA-deficient mice fed a high-fat diet for 6 weeks were investigated for atherosclerotic lesions in vivo. In vitro experiments using RAW264.7 macrophages, primary resident peritoneal macrophages extracted from wild-type or SPA-deficient mice, and human monocyte-derived macrophages from the peripheral blood of healthy donors determined the functional effects of SPA in macrophage foam cell formation. RESULTS: SPA expression was increased in atherosclerotic lesions in humans and ApoE-deficient mice and in response to a proatherosclerotic stimulus in vitro. SPA deficiency reduced the lipid profiles induced by hypercholesterolemia, attenuated atherosclerosis, and reduced the number of lesion-associated macrophage foam cells. In vitro studies revealed that SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA deficiency dramatically downregulated the expression of scavenger receptor CD36 (cluster of differentiation antigen 36) cellular and lesional expression. Importantly, SPA also increased CD36 expression in human monocyte-derived macrophages. CONCLUSIONS: Our results elucidate that SPA is a novel factor promoting atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis by increasing scavenger receptor CD36 expression, leading to increasing cellular OxLDL influx.

C57BL/6 Peritoneal Macrophage Exosomes Improve Antileishmanial Functions of the RAW264.7 Cells.

Leishmaniasis is considered one of the most critical health concerns in the world. Unfortunately, no protective vaccines exist and conventional treatments are relatively ineffective. Therefore, new strategies are necessary against leishmaniasis. In recent years, exosomes have shown promising therapeutic outcomes in various diseases, including infectious diseases. In this regard, we aimed to explore the effect of the exosome, pyrimethamine and their combination on the anti-parasitic function of RAW264.7 cells against Leishmania major. Exosomes were isolated from the C57BL/6 peritoneal macrophages. L. major infected and non-infected RAW264.7 cells treated with exosomes, pyrimethamine (PM), and exosomes along with PM. The effect of the treatments was analysed on phagocytosis, efferocytosis, the intracellular parasite count, arginase activity, nitric oxide (NO) and reactive oxygen species (ROS) production. Exosomes could significantly elevate the phagocytosis, efferocytosis, NO and ROS in both infected and non-infected groups (Pv < 0.05). The exosomes reduced the arginase activity in both groups (Pv < 0.05). The intracellular parasite count was significantly lower after treatment with exosomes (Pv < 0.05). These results demonstrate that MQ-derived exosomes can enhance in vitro anti-parasitic responses against L. major. This provides a potential pathway for more effective treatments and underscores the importance of further research in this area.

Investigating the role and regulation of GPNMB in progranulin-deficient macrophages.

Introduction: Progranulin (PGRN) is a holoprotein that is internalized and taken to the lysosome where it is processed to individual granulins (GRNs). PGRN is critical for successful aging, and insufficient levels of PGRN are associated with increased risk for developing neurodegenerative diseases like AD, PD, and FTD. A unifying feature among these diseases is dysregulation of peripheral immune cell populations. However, considerable gaps exist in our understanding of the function(s) of PGRN/GRNs in immune cells and their role in regulating central-peripheral neuroimmune crosstalk. One of the most upregulated genes and proteins in humans with GRN haploinsufficiency and in aged Grn knock-out (KO) mice is glycoprotein non-metastatic B (GPNMB) but its normal role within the context of immune crosstalk has not been elucidated. Methods: To address this gap, peritoneal macrophages (pMacs) from 5-to-6-month old WT and Grn KO mice were assessed for Gpnmb expression and stimulation-dependent cytokine release in the presence or absence of the Gpnmb extracellular domain (ECD). Cellular localization, as well as inhibition of, the microphthalmia-associated transcription factor (MITF) was assessed to determine its mechanistic role in Gpnmb overexpression in Grn KO pMacs. Results: We observed an increase in GPNMB protein and mRNA as a result of insufficient progranulin in peripheral immune cells at a very early age relative to previous reports on the brain. Stimulation-dependent cytokine release was decreased in the media of Grn KO pMacs relative to WT controls; a phenotype that could be mimicked in WT pMacs with the addition og GPNMB ECD. We also found that MITF is dysregulated in Grn KO pMacs; however, its nuclear translocation and activity are not required to rescue the immune dysregulation of Grn KO macrophages, suggesting redundancy in the system. Discussion: These findings highlight the fact that knowledge of early-stage disease mechanism(s) in peripheral populations may inform treatment strategies to delay disease progression at an early, prodromal timepoint prior to development of neuroinflammation and CNS pathology.

3-Alkoxy-1-Benzyl-5-Nitroindazole Derivatives Are Potent Antileishmanial Compounds.

Indazoles have previously been identified as molecules with antiprotozoal activity. In this study, we evaluate the in vitro activity of thirteen 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) against L. amazonensis, L. infantum, and L. mexicana. In vitro, cytotoxicity against mouse peritoneal macrophages and growth inhibitory activity in promastigotes were evaluated for all compounds, and those showing adequate activity and selectivity were tested against intracellular amastigotes. Transmission and scanning electron microscopy were employed to study the effects of 3-alkoxy-1-benzyl-5-nitroindazole and 2-benzyl-5-nitroindazolin-3-one derivatives on promastigotes of L. amazonensis. Compounds NV6 and NV8 were active in the two life stages of the three species, with the latter showing the best indicators of activity and selectivity. 3-alkoxy-1-benzyl-5-nitroindazole derivatives (series D) showed in vitro activity comparable to that of amphotericin B against the promastigote stage of Leishmania spp. Two compounds were also found to be active the amastigote stage. Electron microscopy studies confirmed the antileishmanial activity of the indazole derivatives studied and support future research on this family of compounds as antileishmanial agents.

Glutamic-Alanine Rich Glycoprotein from Undaria pinnatifida: A Promising Natural Anti-Inflammatory Agent.

This study aimed to assess the anti-inflammatory properties of a bioactive glutamic-alanine rich glycoprotein (GP) derived from Undaria pinnatifida on both LPS-stimulated RAW264.7 cells, peritoneal macrophages, and mouse models of carrageenan- and xylene-induced inflammation, investigating the underlying molecular mechanisms. In both in-vitro and in-vivo settings, GP was found to reduce the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) while also inhibiting the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in response to lipopolysaccharide (LPS) stimulation. GP treatment significantly impeded the nuclear translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway by blocking the phosphorylation of IKKα and IκBα, leading to a reduction in proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Additionally, GP effectively inhibited the activation of mitogen-activated protein kinases (MAPKs), with specific inhibitors of p38 and extra-cellular signal regulated kinase (ERK) enhancing GP's anti-inflammatory efficacy. Notably, GP administration at 10 mg/kg/day (p.o.) markedly reduced carrageenan-induced paw inflammation and xylene-induced ear edema by preventing the infiltration of inflammatory cells into targeted tissues. GP treatment also downregulated key inflammatory markers, including iNOS, COX-2, IκBα, and NF-κB, by suppressing the phosphorylation of p38 and ERK, thereby improving the inflammatory index in both carrageenan- and xylene-induced mouse models. These findings suggest that marine resources, particularly seaweeds like U. pinnatifida, could serve as valuable sources of natural anti-inflammatory proteins for the effective treatment of inflammation and related conditions.

Neu1 deficiency and fibrotic lymph node microenvironment lead to imbalance in M1/M2 macrophage polarization.

Macrophages play a pivotal role in tissue homeostasis, pathogen defense, and inflammation resolution. M1 and M2 macrophage phenotypes represent two faces in a spectrum of responses to microenvironmental changes, crucial in both physiological and pathological conditions. Neuraminidase 1 (Neu1), a lysosomal and cell surface sialidase responsible for removing terminal sialic acid residues from glycoconjugates, modulates several macrophage functions, including phagocytosis and Toll-like receptor (TLR) signaling. Current evidence suggests that Neu1 expression influences M1/M2 macrophage phenotype alterations in the context of cardiovascular diseases, indicating a potential role for Neu1 in macrophage polarization. For this reason, we investigated the impact of Neu1 deficiency on macrophage polarization in vitro and in vivo. Using bone marrow-derived macrophages (BMDMs) and peritoneal macrophages from Neu1 knockout (Neu1-/- ) mice and wild-type (WT) littermate controls, we demonstrated that Neu1-deficient macrophages exhibit an aberrant M2-like phenotype, characterized by elevated macrophage mannose receptor 1 (MMR/CD206) expression and reduced responsiveness to M1 stimuli. This M2-like phenotype was also observed in vivo in peritoneal and splenic macrophages. However, lymph node (LN) macrophages from Neu1-/- mice exhibited phenotypic alterations with reduced CD206 expression. Further analysis revealed that peripheral LNs from Neu1-/- mice were highly fibrotic, with overexpression of transforming growth factor-beta 1 (TGF-ß1) and hyperactivated TGF-ß signaling in LN macrophages. Consistently, TGF-ß1 was found to alter M1/M2 macrophage polarization in vitro. Our findings showed that Neu1 deficiency prompts macrophages towards an M2 phenotype and that microenvironmental changes, particularly increased TGF-ß1 in fibrotic tissues such as peripheral LNs in Neu1-/- mice, further influence M1/M2 macrophage polarization, highlighting its sensitivity to the local microenvironment. Therapeutic interventions targeting Neu1 or TGF-ß signaling pathways may offer the potential to regulate macrophage behavior across different diseases.

Sprayable anti-adhesive hydrogel for peritoneal macrophage scavenging in post-surgical applications.

Post-surgical adhesions frequently occur after intra-abdominal surgery, leading to severe complications. Despite the development of various types of adhesion barriers to address post-surgical adhesions, several limitations persist, including off-target localization, handling difficulties, and potential immunogenicity. Here, we report a spray-type adhesion barrier for broad, fast application, forming two sequential networks. The first network is formed by a polyelectrolyte complex of sulfated hyaluronic acid and chitosan, while the second network is established through pluronic® F127 thermogelation. This sprayable barrier served as both a physical protector for the damaged peritoneum and an immunomodulator for peritoneal macrophages, as evidenced its effectiveness in a rat ischemic button model. Taken together, this efficient adhesion barrier presents a promising solution for post-surgical adhesions.

Both host and parasite non-coding RNAs co-ordinate the regulation of macrophage gene expression to reduce pro-inflammatory immune responses and promote tissue repair pathways during infection with fasciola hepatica.

Parasitic worms (helminths) establish chronic infection within mammalian hosts by strategically regulating their host's immune responses. Deciphering the mechanisms by which host non-coding RNAs (ncRNA) co-ordinate the activation and regulation of immune cells is essential to understanding host immunity and immune-related pathology. It is also important to comprehend how pathogens secrete specific ncRNAs to manipulate gene expression of host immune cells and influence their response to infection. To investigate the contribution of both host and helminth derived ncRNAs to the activation and/or regulation of innate immune responses during a parasite infection, we examined ncRNA expression in the peritoneal macrophages from mice infected with Fasciola hepatica. We discovered the presence of several parasitic-derived miRNAs within host macrophages at 6 hrs and 18 hrs post infection. Target prediction analysis showed that these Fasciola miRNAs regulate host genes associated with the activation of host pro-inflammatory macrophages. Concomitantly, there was a distinct shift in host ncRNA expression, which was significant at 5 days post-infection. Prediction analysis suggested that these host ncRNAs target a different cohort of host genes compared to the parasite miRNAs, although the functional outcome was predicted to be similar i.e. reduced pro-inflammatory response and the promotion of a reparative/tolerant phenotype. Taken together, these observations uncover the interplay between host and parasitic ncRNAs and reveal a complementary regulation of the immune response that allows the parasite to evade immune detection and promote tissue repair for the host. These findings will provide a new understanding of the molecular interaction between parasites and host.

Immunomodulatory Effects and Regulatory Mechanisms of (R)-6-HITC, an Isothiocyanate from Wasabi (Eutrema japonicum), in an Ex Vivo Mouse Model of LPS-Induced Inflammation.

The present study aimed to investigate the effects of (R)-(-)-1-isothiocyanato-6-(methylsulfinyl)-hexane [(R)-6-HITC], the major isothiocyanate present in wasabi, in an ex vivo model of inflammation using lipopolysaccharide-stimulated murine peritoneal macrophages. (R)-6-HITC improved the immune response and mitigated oxidative stress, which involved suppression of reactive oxygen species, nitric oxide, and pro-inflammatory cytokines (IL-1ß, IL-6, IL-17, IL-18, and TNF-α) production and downregulation of pro-inflammatory enzymes such as inducible nitric oxide synthase, COX-2, and mPGES-1. In addition, (R)-6-HITC was able to activate the Nrf2/HO-1 axis while simultaneously inhibiting key signaling pathways, including JAK2/STAT3, mitogen-activated protein kinases, and canonical and noncanonical inflammasome pathways, orchestrating its potent immunomodulatory effects. Collectively, these findings demonstrate the potential of (R)-6-HITC as a promising nutraceutical for the management of immuno-inflammatory diseases and justify the need for further in vivo validation studies.

Comparison of structural characteristics and anti-tumor activity of two alkali extracted peach gum arabinogalactan.

Two polysaccharides, PGP-90 and PGP-100 (molecular weights of 7.59 × 102 kDa and 10.48 × 102 kDa, respectively), were isolated from Peach gum using alkaline electrolyte water as an extraction solution. Structural characterization showed that PGP-90 and PGP-100 are AG-II arabinogalactans with ß-D-(1 â†’ 6)-Galp as the main chain and 1 â†’ 3 Araf and 1 â†’ 5 Araf branched chains at O-3 and O-4 positions. Animal experiments showed that PGP-90 and PGP-100 significantly improved immune function, enhance the proliferative capacity of lymphocytes and phagocytosis of peritoneal macrophages, and regulated the ratio of lymphocyte subpopulations in S180 tumor-bearing mice. Meanwhile, PGP-90 and PGP-100 promoted the secretion of cytokines (TNF-α, IFN-γ, and IL-2) by activated macrophages and blocked apoptosis at the G1 phase, resulting in tumor suppression rates of 40.80 % and 46.30 % (100 mg/kg), respectively, with PGP-100 demonstrating stronger in vivo anti-tumor activity. The above experimental results indicate that Peach gum polysaccharides have the potential to be functional anti-tumor agents.

Broad-spectrum antibiotics disrupt homeostatic efferocytosis.

The clearance of apoptotic cells, termed efferocytosis, is essential for tissue homeostasis and prevention of autoimmunity1. Although past studies have elucidated local molecular signals that regulate homeostatic efferocytosis in a tissue2,3, whether signals arising distally also regulate homeostatic efferocytosis remains elusive. Here, we show that large peritoneal macrophage (LPM) display impairs efferocytosis in broad-spectrum antibiotics (ABX)-treated, vancomycin-treated and germ-free mice in vivo, all of which have a depleted gut microbiota. Mechanistically, the microbiota-derived short-chain fatty acid butyrate directly boosts efferocytosis efficiency and capacity in mouse and human macrophages, and rescues ABX-induced LPM efferocytosis defects in vivo. Bulk messenger RNA sequencing of butyrate-treated macrophages in vitro and single-cell messenger RNA sequencing of LPMs isolated from ABX-treated and butyrate-rescued mice reveals regulation of efferocytosis-supportive transcriptional programmes. Specifically, we find that the efferocytosis receptor T cell immunoglobulin and mucin domain containing 4 (TIM-4, Timd4) is downregulated in LPMs of ABX-treated mice but rescued by oral butyrate. We show that TIM-4 is required for the butyrate-induced enhancement of LPM efferocytosis capacity and that LPM efferocytosis is impaired beyond withdrawal of ABX. ABX-treated mice exhibit significantly worse disease in a mouse model of lupus. Our results demonstrate that homeostatic efferocytosis relies on distal metabolic signals and suggest that defective homeostatic efferocytosis may explain the link between ABX use and inflammatory disease4-7.

Exotoxins secreted by Clostridium septicum induce macrophage death: Implications for bacterial immune evasion mechanisms at infection sites.

The induction of macrophage death is considered a potential mechanism by which components secreted by Clostridium septicum are used to evade the innate immune response and cause tissue damage. This study aimed to determine the effects of partially purified fractions of extracellular proteins secreted by C. septicum on the death of mouse peritoneal macrophages. Elicited mouse peritoneal macrophages were incubated with partially purified fractions of proteins secreted by C. septicum into the culture medium. After incubation, the protein fraction with a molecular weight ≥100 kDa caused significant cell death in macrophages, altered cell morphology, increased the expression of markers of apoptosis and autophagy, and increased the expression (protein and mRNA) of IL-10 and TNFα. Our data suggest that the proteins secreted by C. septicum (MW, ≥100 kDa) induce cell death in macrophages by promoting autophagy-triggered apoptosis. This study may contribute to our understanding of the molecular mechanism of immune evasion by C. septicum at the infection site.

Microemulsions strongly promoted the activity of α-bisabolol against different Leishmania species and its skin permeation.

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials.

Antileishmanial activity of a dillapiole derivative obtained from Piper aduncum L. (Piperaceae).

Cutaneous leishmaniasis (CL) is considered a public health problem. Current treatments have disadvantages because they are invasive and have serious side effects, and thus there is a need for research into new, more effective pharmacological alternatives. Plants are promising sources of bioactive substances, and new analogues can be obtained through chemical reactions. The present study aimed to evaluate the antileishmanial effects of the analog dillapiole n-butyl ether (DBE) extracted from Piper aduncum leaves. The cytotoxic potential of DBE was evaluated at concentrations of 15.62 to 500 µM in peritoneal macrophages for 48 h, and in RAW 264.7 macrophages for 72 h using a dose-response method. The antileishmanial activity in L. amazonensis promastigotes used concentrations of 0.2 to 4.5 µM for 24, 48 and 72 h and the quantification of the cellular infection rate used a concentration of 4.5 µM of DBE against the amastigote forms internalized in macrophages for 24 h and 48 h. Nitric oxide was quantified from macrophages previously treated with DBE for 24 h and 48 h. The dosage of reactive oxygen species used a concentration of 4.5 µM of DBE incubated together with dichlorofluorescein acetate for 1, 3, 6, and 24 h. For the molecular modeling of DBE, the Leishmania protein, available in the "Protein Data Bank" database, was used. The studied molecule was not toxic to cells and presented a CC50 of 413 µM in peritoneal macrophages and 373.5 µM in RAW 264.7. The analogue inhibited promastigote forms of L. amazonensis with an IC50 of 1.6 µM for 72 h. DBE presented an infection rate of 17% and 12%, dillapiole of 24% and 14% and Pentacarinat® of 10% and 9% over 48 h. DBE demonstrated a binding energy of -7.8 for the U53 enzyme. It is concluded that the analogue showed promising antileishmanial activity for future in vivo tests.

The involvement of peritoneal GATA6+ macrophages in the pathogenesis of endometriosis.

Endometriosis is a chronic inflammatory disease that causes debilitating pelvic pain in women. Macrophages are considered to be key players in promoting disease progression, as abundant macrophages are present in ectopic lesions and elevated in the peritoneum. In the present study, we examined the role of GATA6+ peritoneal macrophages on endometriosis-associated hyperalgesia using mice with a specific myeloid deficiency of GATA6. Lesion induction induced the disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased Ly6C+ monocytes and TIM4lo MHCIIhi macrophages. The recruitment of MHCIIhi inflammatory macrophages was extensive in Mac Gata6 KO mice due to the severe disappearance of TIM4hi MHCIIlo residential macrophages. Ki67 expression confirmed GATA6-dependent proliferative ability, showing different proliferative phenotypes of TIM4+ residential macrophages in Gata6f/f and Mac Gata6 KO mice. Peritoneal proinflammatory cytokines were elevated after lesion induction. When cytokine levels were compared between Gata6f/f and Mac Gata6 KO mice, TNFα at day 21 in Gata6f/f mice was higher than in Mac Gata6 KO mice. Lesion induction increased both abdominal and hind paw sensitivities. Gata6f/f mice tended to show higher sensitivity in the abdomen after day 21. Elevated expression of TRPV1 and CGRP was observed in the dorsal root ganglia after ELL induction in Gata6f/f mice until days 21 and 42, respectively. These results support that peritoneal GATA6+ macrophages are involved in the recruitment and reprogramming of monocyte-derived macrophages. The extensive recruitment of monocyte-derived macrophages in Mac Gata6 KO mice might protect against inflammatory stimuli during the resolution phase, whereas GATA6 deficiency did not affect lesion initiation and establishment at the acute phase of inflammation. GATA6+ residential macrophages act to sustain local inflammation in the peritoneum and sensitivities in the neurons, reflecting endometriosis-associated hyperalgesia.

The suppressive role of NLRP6 in host defense against Streptococcus suis infection.

Streptococcus suis (S. suis) disease is a prevalent zoonotic infectious threat that elicits a systemic inflammatory response in both swine and humans, frequently culminating in high mortality rates. The excessive inflammation triggered by S. suis infection can precipitate tissue damage and sudden death; however, a comprehensive strategy to mitigate this inflammatory response remains elusive. Our study examines the role of NLRP6 in S. suis infection, with a particular focus on its involvement in pathogen regulation. A marked upregulation of NLRP6 was observed in peritoneal macrophages post-infection with S. suis SC19 strain, consequently activating the NLRP6 inflammasome. Furthermore, SC19 infection was found to augment the secretion of pro-inflammatory cytokines IL-1ß via NLRP6 activation, while NLRP6 deficiency mitigates the invasion and adhesion of SC19 to macrophages. In vivo models revealed that NLRP6 deletion enhanced survival rates of SC19-infected mice, alongside a reduction in tissue bacterial load and inflammatory cytokine levels. NLRP6-/- mice were shown to exhibit attenuated inflammatory responses in pulmonary, hepatic, and splenic tissues post-SC19 infection, as evidenced by lower inflammation scores. Flow cytometry analyses further substantiated that NLRP6 is involved in modulating macrophage and neutrophil recruitment during infection. Our findings suggest that NLRP6 negatively regulates host resistance against S. suis infection; its absence results in reduced mortality, bacterial colonization, and a milder inflammatory response. Elucidating the mechanism of NLRP6 in S. suis-induced inflammation provides novel insights and theoretical underpinnings for the prophylaxis and therapeutics of S. suis diseases.

Effect of Modified Forms of Sodium Humate PowHumus on the Balance of Arginine in Peritoneal Macrophages of Intact Mice.

Substances of silver nanoparticles dialyzed through a 13 kDa membrane, synthesized in a medium of humic ligands modified with hydroquinone and 2-hydroxynaphthoquinone from PowHumus brown coal, specifically enhance the M2 properties of peritoneal macrophages due to inhibition of NO synthase and significant activation of arginase, thus enhancing anti-inflammatory properties of cells. In small, but effective concentrations, they do not have cytotoxic properties and do not contain pyrogenic impurities. The studied humates are able to influence the mechanisms of immune response formation and are an effective means for correcting inflammation and regeneration.

Immunoneuroendocrine, Stress, Metabolic, and Behavioural Responses in High-Fat Diet-Induced Obesity.

Obesity has reached global epidemic proportions, and even though its effects are well-documented, studying the interactions among all influencing factors is crucial for a better understanding of its physiopathology. In a high-fat-diet-induced obesity animal model using C57BL/6J mice, behavioural responses were assessed through a battery of tests, while stress biomarkers and systemic inflammatory cytokines were measured using an Enzyme-Linked ImmunoSorbent Assay and a Bio-Plex Multiplex System. The peritoneal macrophage microbicide capacity was analysed via flow cytometry, and crown-like structures (CLSs) in white adipose tissue (WAT) were evaluated through staining techniques. Results indicated that obese mice exhibited increased body weight, hyperglycaemia, and hyperlipidaemia after 18 weeks on a high-fat diet, as well as worse physical conditions, poorer coordination and balance, and anxiety-like behaviour. Differences in corticosterone and noradrenaline concentrations were also found in obese animals, revealing a stress response and noradrenergic dysregulation, along with a weakened innate immune response characterized by a lower microbicide capacity, and the presence of an underlying inflammation evidenced by more CLSs in WAT. Altogether, these findings indicate that obesity deteriorates the entire stress, inflammatory, metabolic, sensorimotor and anxiety-like behavioural axis. This demonstrates that jointly evaluating all these aspects allows for a deeper and better exploration of this disease and its associated comorbidities, emphasizing the need for individualized and context-specific strategies for its management.