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Rheumatoid arthritis (RA) is a chronic inflammatory disease that eventually leads to disability. Inflammatory cell infiltration, severe joint breaking and systemic bone loss are the main clinical symptoms. In this study, we established a collagen-induced arthritis (CIA) model and found a large number of M1 macrophages and pyroptosis, which are important sources of proinflammatory cytokines. Punicalagin (PUN) is an active substance extracted from pomegranate peel. We found that it inhibited joint inflammation, cartilage damage and systemic bone destruction in CIA mice. PUN effectively alleviated the high expression of inflammatory cytokines in synovial tissue in vivo. PUN treatment shifted macrophages from the M1 phenotype to the M2 phenotype after stimulation with lipopolysaccharide (LPS) and interferon (IFN)-γ. The expression of inducible nitric oxide synthase (iNOS) and other proinflammatory cytokines released by M1 macrophages was decreased in the PUN treatment group. However, simultaneously, the expression of markers of anti-inflammatory M2 macrophages, such as arginase (Arg)-1 and interleukin (IL)-10, was increased. In addition, PUN treatment attenuated pyroptosis by downregulating the expression of NLRP3 and caspase-1, thereby preventing inflammatory cell death resulting from the release of IL-1β and IL-18. Mechanistically, PUN inhibited the activation of receptor activators of the nuclear factor-κB (NF-κB) signaling pathway, which contributes to M1 polarization and pyroptosis of macrophages. We concluded that PUN ameliorated pathological inflammation by inhibiting M1 phenotype polarization and pyroptosis and has great potential as a therapeutic treatment for human RA., (© 2021. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Involvement of macrophages in the SARS-CoV-2-associated cytokine storm, the excessive secretion of inflammatory/anti-viral factors leading to the acute respiratory distress syndrome (ARDS) in COVID-19 patients, is unclear. In this study, we sought to characterize the interplay between the virus and primary human monocyte-derived macrophages (MDM). MDM were stimulated with recombinant IFN-α and/or infected with either live or UV-inactivated SARS-CoV-2 or with two reassortant influenza viruses containing external genes from the H1N1 PR8 strain and heterologous internal genes from a highly pathogenic avian H5N1 or a low pathogenic human seasonal H1N1 strain. Virus replication was monitored by qRT-PCR for the E viral gene for SARS-CoV-2 or M gene for influenza and TCID 50 or plaque assay, and cytokine levels were assessed semiquantitatively with qRT-PCR and a proteome cytokine array. We report that MDM are not susceptible to SARS-CoV-2 whereas both influenza viruses replicated in MDM, albeit abortively. We observed a modest cytokine response in SARS-CoV-2 exposed MDM with notable absence of IFN-β induction, which was instead strongly induced by the influenza viruses. Pre-treatment of MDM with IFN-α enhanced proinflammatory cytokine expression upon exposure to virus. Together, the findings concur that the hyperinflammation observed in SARS-CoV-2 infection is not driven by macrophages.

Although skeletal muscle is the main effector organ largely accounting for disability after stroke, considerably less attention is paid to the secondary abnormalities of stroke-related skeletal muscle loss. It is necessary to explore the mechanism of muscle atrophy after stroke and further develop effective rehabilitation strategy. Here, we evaluated the effects of high-intensity interval (HIIT) versus moderate-intensity aerobic training (MOD) on physical function, muscle mass, and stroke-related gene expression profile of skeletal muscle. After the model of middle cerebral artery occlusion (MCAO) was successfully made, the blood lactate threshold corresponding speed ( S LT ) and maximum speed ( S max ) were measured. Different intensity training protocols (MOD < S LT and IL-6 in paretic gastrocnemius, and improving peripheral blood CD4+/CD8+ T cell ratio, level of IL-10. Additionally, RNA-seq analysis revealed that the differentially expressed genes among HIIT, MOD, and MCAO groups were highly associated with signaling pathways involved in inflammatory response, more specifically the I-kappaB kinase/NF-kappaB signaling. Following the outcome, we further investigated the infiltrating immune cells abundant in paretic muscles. The results showed that HIIT modulated macrophage activation by downregulating CD86+ (M1 type) macrophages and upregulating CD163+ (M2 type) macrophages via inhibiting the TLR4/MyD88/NF S LT < HIIT < S max ) were carried out for 3 weeks beginning at 7 days after MCAO in the MOD and HIIT groups, respectively. We found that both HIIT and MOD prevented stroke-related gastrocnemius muscle mass loss in MCAO mice. HIIT was more beneficial than MOD for improvements in muscle strength, motor coordination, walking competency, and cardiorespiratory fitness. Furthermore, HIIT was superior to MOD in terms of reducing lipid accumulation, levels of IL-1 β and IL-6 in paretic gastrocnemius, and improving peripheral blood CD4+/CD8+ T cell ratio, level of IL-10. Additionally, RNA-seq analysis revealed that the differentially expressed genes among HIIT, MOD, and MCAO groups were highly associated with signaling pathways involved in inflammatory response, more specifically the I-kappaB kinase/NF-kappaB signaling. Following the outcome, we further investigated the infiltrating immune cells abundant in paretic muscles. The results showed that HIIT modulated macrophage activation by downregulating CD86+ (M1 type) macrophages and upregulating CD163+ (M2 type) macrophages via inhibiting the TLR4/MyD88/NF κ B signaling pathway and exerting an anti-inflammatory effect in paretic skeletal muscle. It is expected that these data will provide novel insights into the mechanisms and potential targets underlying muscle wasting in stroke., Competing Interests: The authors declare no competing or financial interests., (Copyright © 2021 Lu Luo et al.)

The influenza virus infection poses a serious health threat worldwide. Myeloid cells play pivotal roles in regulating innate and adaptive immune defense. A disintegrin and metalloproteinase (ADAM) family of proteins contributes to various immune responses; however, the role of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in influenza virus infection remains largely unknown. Herein, we investigated its role, focusing on myeloid cells, during influenza virus infection in mice. ADAM10 gene ( Adam10 ) flox/flox /Lyz2-Cre ( Adam10 ΔLyz2 ) and control Adam10 flox/flox mice were intranasally infected with 200 plaque-forming units of influenza virus A/H1N1/PR8/34. Adam10 ΔLyz2 mice exhibited a significantly higher mortality rate, stronger lung inflammation, and a higher virus titer in the lungs than control mice. Macrophages and inflammatory cytokines, such as TNF-α, IL-1β, and CCL2, were increased in bronchoalveolar lavage fluid from Adam10 ΔLyz2 mice following infection. CD11b + Ly6G - F4/80 + myeloid cells, which had an inflammatory monocyte/macrophage-like phenotype, were significantly increased in the lungs of Adam10 ΔLyz2 mice. Adoptive transfer experiments suggested that these cells likely contributed to the poorer prognosis in Adam10 ΔLyz2 mice. Seven days after infection, CD11b + Ly6G - F4/80 + lung cells exhibited significantly higher arginase-1 expression levels in Adam10 ΔLyz2 mice than in control mice, whereas an arginase-1 inhibitor improved the prognosis of Adam10 ΔLyz2 mice. Enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF)/GM-CSF receptor signaling likely contributed to this process. Collectively, these results indicate that myeloid ADAM10 protects against influenza virus pneumonia and may be a promising therapeutic target.

We aimed to investigate the effects of ethanol and its metabolites (β-hydroxybutyrate and sodium acetate) in the effector functions of macrophages in response to Paracoccidioides brasiliensis yeast cells and to determine their influence in the development of the adaptive response. Purified peripheral blood monocytes were differentiated into macrophages and were treated with ethanol, β-hydroxybutyrate, and sodium acetate, and stimulated with P. brasiliensis yeast cells and evaluated for their phenotypic characteristics, functional activity, and capability to induce T cells activation/differentiation. We found that the ethanol treatment diminished the expression of HLA-AB, HLA-DR, CD80, and CD86, modulating the expression of dectin-1, as well as Syk phosphorylation. The ethanol treatment increased the phagocytic activity, expression of CD206, and IL-10 production; however, reduced ROS production, fungicidal activity, caspase-1 cleavage, and IL-1β and IL-6 production. Our data also showed that the presence of ethanol reduced the differentiation of Th1 and Th17 cells and increased the frequency of Th2 cells. Our results indicated that ethanol exposure could suppress effector function of macrophages, possibly leading to the polarization of M2 macrophages. The ethanol modulates the expression of costimulatory and antigen-presentation molecules and interferes with the NLRP3 inflammasome. Altogether, these alterations affect the development of the adaptive response, decreasing the frequency of IL-17, IL-22, and IFN- γ producing cells, and increasing the frequency of IL-4 producing cells. Therefore, exposure to ethanol can impair the capability of macrophages to exert their effector functions and activate the acquired response related to resistance to P. brasiliensis infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 nondiabetic adults across a range of body mass indexes (BMI 20.5-45.8 kg/m 2 ). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (S I ) and β cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, S I , and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced S I , and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with S I , and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements. NEW AND NOTEWORTHY Adipose inflammation is proposed to be at the nexus of the systemic inflammation and metabolic derangements associated with obesity. The present study provides evidence to support adipose inflammation as a central feature of the pathophysiology of obesity. Adipose inflammation is associated with systemic and peripheral metabolic derangements, including increased systemic inflammation, reduced insulin sensitivity, and reduced skeletal muscle mitochondrial respiration.

Activation of immune cells in response to fungal infection involves the reprogramming of their cellular metabolism to support antimicrobial effector functions. Although metabolic pathways such as glycolysis are known to represent critical regulatory nodes in antifungal immunity, it remains undetermined whether these are differentially regulated at the interindividual level. In this study, we identify a key role for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in the immunometabolic responses to Aspergillus fumigatus. A genetic association study performed in 439 recipients of allogeneic hematopoietic stem cell transplantation (HSCT) and corresponding donors revealed that the donor, but not recipient, rs646564 variant in the PFKFB3 gene increased the risk of invasive pulmonary aspergillosis (IPA) after transplantation. The risk genotype impaired the expression of PFKFB3 by human macrophages in response to fungal infection, which was correlated with a defective activation of glycolysis and the ensuing antifungal effector functions. In patients with IPA, the risk genotype was associated with lower concentrations of cytokines in the bronchoalveolar lavage fluid samples. Collectively, these findings demonstrate the important contribution of genetic variation in PFKFB3 to the risk of IPA in patients undergoing HSCT and support its inclusion in prognostic tools to predict the risk of fungal infection in this clinical setting. IMPORTANCE The fungal pathogen Aspergillus fumigatus can cause severe and life-threatening forms of infection in immunocompromised patients. Activation of glycolysis is essential for innate immune cells to mount effective antifungal responses. In this study, we report the contribution of genetic variation in the key glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) to the risk of invasive pulmonary aspergillosis (IPA) after allogeneic hematopoietic stem cell transplantation. The PFKFB3 genotype associated with increased risk of infection was correlated with an impairment of the antifungal effector functions of macrophages in vitro and in patients with IPA. This work highlights the clinical relevance of genetic variation in PFKFB3 to the risk of IPA and supports its integration in risk stratification and preemptive measures for patients at high risk of IPA.

Chemodietary agents are emerging as promising adjuvant therapies in treating various disease conditions. However, there are no adjuvant therapies available to minimize the neurotoxicity of currently existing antiretroviral drugs (ARVs). In this study, we investigated the anti-HIV effect of a chemodietary agent, Cucurbitacin-D (Cur-D), in HIV-infected macrophages using an in-vitro blood-brain barrier (BBB) model. Since tobacco smoking is prevalent in the HIV population, and it exacerbates HIV replication, we also tested the effect of Cur-D against cigarette smoke condensate (CSC)-induced HIV replication. Our results showed that Cur-D treatment reduces the viral load in a dose-dependent (0-1 μM) manner without causing significant toxicity at <1 μM concentration. Further, a daily dose of Cur-D (0.1 μM) not only reduced p24 in control conditions, but also reduced CSC (10 μg/mL)-induced p24 in U1 cells. Similarly, Cur-D (single dose of 0.4 μM) significantly reduced the CSC (single dose of 40 μg/mL)-induced HIV replication across the BBB model. In addition, treatment with Cur-D reduced the level of pro-inflammatory cytokine IL-1β. Therefore, Cur-D, as an adjuvant therapy, may be used not only to suppress HIV in the brain, but also to reduce the CNS toxicity of currently existing ARVs.

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-β) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. IMPORTANCE Streptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes , on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response., (Copyright © 2021 Rahbari et al.)

Macrophages play a major role in innate immune responses by producing a variety of immune mediators and cytokines. The stimulation of macrophages by natural products may lead to an enhanced innate immune system. This study evaluated the immunostimulatory effects of a polysaccharide-rich crude fraction of Celosia cristata L. flowers (CCP) on murine macrophages. CCP treatment induced the production of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines by macrophages. Mechanistically, the activation of mitogen-activated protein kinases, NF-κB and toll-like receptor 4 were found to be associated with the stimulatory functions of CCP. CCP was found to be primarily composed of galacturonic acid and glucose in addition to small amounts of arabinose and galactose. This study demonstrated that CCP may enhance the innate immune responses and potentially improve the immune functions in the body., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

Introduction. Macrophages polarization is essential in infection control. Llipopolysaccharide (LPS) plays an essential role in host innate immune system-pathogen interaction. The LPS structure of Pseudomonas aeruginosa modifies in the adaptation of this pathogen to biofilm-related chronic infection. Gap statement. There have been several studies on LPS induced polarization of human and mouse macrophages with different results. And it was reported that the lipid A structure of the LPS derived from biofilm-forming Pseudomonas aeruginosa strain PAO1 was modified. Aim. This study aimed to investigate the effect and the involved pathway of LPS from biofilm-forming PAO1 on human and murine macrophage polarization. Methodology. LPS was isolated from biofilm-forming and planktonic PAO1 and quantified. Then the LPS was added to PMA-differentiated human macrophage THP-1 cells and Raw264.7 murine macrophage cells. The expression of iNOS, Arg-1, IL4 , TNF-α, CCL3 , and CCL22 was analysed in the different cell lines. The expression of TICAM-1 and MyD88 in human THP-1 macrophages was quantified by Western blot. PAO1 infected macrophages at different polarization states, and the intracellular bacterial growth in macrophages was evaluated. Results. LPS from biofilm-forming PAO1 induced more marked hyperinflammatory responses in THP-1 and Raw264.7 macrophages than LPS derived from planktonic PAO1, and these responses were related to the up-regulation of MyD88. Intracellular growth of PAO1 was significantly increased in THP-1 macrophages polarized by LPS from biofilm-forming PAO1, but decreased both in THP-1 and Raw264.7 macrophages polarized by LPS from planktonic PAO1. Conclusion. The presented in vitro study indicates that LPS derived from biofilm-forming PAO1 induces enhanced M1 polarization in human and murine macrophage cell lines than LPS from planktonic PAO1.

The discovery of opioid receptor expression on immune cells has originated a large research activity on the possible modulation by opioid drugs of immune system responses. In this chapter we describe an easy methodology useful to obtain information about the potential immunomodulatory activity of opioid drugs. An in vivo treatment schedule is used, and macrophages are studied for their ability to release different cytokines.

Staphylococcus aureus (S. aureus) is a gram-positive bacteria, which causes various fatal respiratory infections including pneumonia. The emergence of Methicillin-Resistance Staphylococcus aureus (MRSA) demands a thorough understanding of host-pathogen interactions. Here we report the role of calcium in regulating defence responses of S. aureus in macrophages. Regulating calcium fluxes in cells by different routes differentially governs the expression of T cell costimulatory molecule CD80 and Th1 promoting IL-12 receptor. Inhibiting calcium influx from extracellular medium increased expression of IFN-γ and IL-10 while blocking calcium release from the intracellular stores inhibited TGF-β levels. Blocking voltage-gated calcium channels (VGCC) inhibited the expression of multiple cytokines. While VGCC regulated the expression of apoptosis protein Bax, extracellular calcium-regulated the expression of Cytochrome-C. Similarly, VGCC regulated the expression of autophagy initiator Beclin-1. Blocking VGCC or calcium release from intracellular stores promoted phagosome-lysosome fusion, while activating VGCC inhibited phagosomelysosome fusion. Finally, calcium homeostasis regulated intracellular growth of Staphylococcus, although using different mechanisms. While blocking extracellular calcium influx seems to rely on IFN-γ and IL-12Rβ receptor mediated reduction in bacterial survival, blocking either intracellular calcium release or via VGCC route seem to rely on enhanced autophagy mediated reduction of intracellular bacterial survival. These results point to fine-tuning of defence responses by routes of calcium homeostasis., Competing Interests: Authors state no conflict of interest., (Open Access. © 2020 Chaitenya Verma et al., published by De Gruyter.)

Background: Leishmania braziliensis is prevalent in Latin American countries, including Brazil. It causes cutaneous and mucocutaneous leishmaniasis, leading to high morbidity, and has a low cure rate. Treatment is based on pentavalent antimonials; nonetheless, there are problems related to high toxicity, high cost, and parasitic resistance. Discovery of new leishmanicidal drugs without these limitations and that stimulate the cellular immune response is necessary., Purpose: The present work evaluates whether Astronium fraxinifolium Schott exerts leishmanicidal activity against L. braziliensis by providing a classically polarized profile in infected macrophages., Methods: For the evaluation of the A. fraxinifolium Schott leishmanicidal activity, amastigote cell death was demonstrated in infected RAW 267.4 macrophages treated with an ethanolic extract from the plant sapwood (EEAF). For the evaluation of the EEAF capacity in providing a classically polarized profile in infected macrophages, the following analyses were done: detection of LAMP-1 protein by the baculovirus technology, measurement of superoxide anion by the NBT testing, quantification of TNF-α, IL-12p40, IL-10, IL-4, and TGF-β by sandwich-type enzyme immune assays, and iNOS and COX-2 expression by RT-PCR technique., Results: The EEAF significantly reduced amastigote counts inside the cells. Vacuoles were visualized in infected and treated cells before and after May-Grünwald-Giemsa staining. A strong LAMP-1 protein fluorescence revealed phagosome maturation in infected cells treated with the EEAF. No production of superoxide was visualized in infected cells treated with the plant material. Nonetheless, high levels of TNF-α, IL-12p40, and IL-10 were found in cell supernatants, but reduced levels of TGF-β and no IL-4 production. We identified augmented mRNA expression for COX-2, but no expression of iNOS mRNA., Conclusion: Our results demonstrated that A. fraxinifolium induced a classically polarized profile in infected macrophages but also provided a less harmful environment by stimulating the production of certain anti-inflammatory mediators, such as IL-10.

Macrophage activation involves metabolic reprogramming to support antimicrobial cellular functions. How these metabolic shifts influence the outcome of infection by intracellular pathogens remains incompletely understood. Mycobacterium tuberculosis (Mtb) modulates host metabolic pathways and utilizes host nutrients, including cholesterol and fatty acids, to survive within macrophages. We found that intracellular growth of Mtb depends on host fatty acid catabolism: when host fatty acid β-oxidation (FAO) was blocked chemically with trimetazidine, a compound in clinical use, or genetically by deletion of the mitochondrial fatty acid transporter carnitine palmitoyltransferase 2 (CPT2), Mtb failed to grow in macrophages, and its growth was attenuated in mice. Mechanistic studies support a model in which inhibition of FAO generates mitochondrial reactive oxygen species, which enhance macrophage NADPH oxidase and xenophagy activity to better control Mtb infection. Thus, FAO inhibition promotes key antimicrobial functions of macrophages and overcomes immune evasion mechanisms of Mtb. IMPORTANCE (Mtb) is the leading infectious disease killer worldwide. We discovered that intracellular Mtb fails to grow in macrophages in which fatty acid β-oxidation (FAO) is blocked. Macrophages treated with FAO inhibitors rapidly generate a burst of mitochondria-derived reactive oxygen species, which promotes NADPH oxidase recruitment and autophagy to limit the growth of Mtb. Furthermore, we demonstrate the ability of trimetazidine to reduce pathogen burden in mice infected with Mtb. These studies will add to the knowledge of how host metabolism modulates Mtb infection outcomes.Mycobacterium tuberculosis (Mtb) is the leading infectious disease killer worldwide. We discovered that intracellular Mtb fails to grow in macrophages in which fatty acid β-oxidation (FAO) is blocked. Macrophages treated with FAO inhibitors rapidly generate a burst of mitochondria-derived reactive oxygen species, which promotes NADPH oxidase recruitment and autophagy to limit the growth of Mtb. Furthermore, we demonstrate the ability of trimetazidine to reduce pathogen burden in mice infected with Mtb. These studies will add to the knowledge of how host metabolism modulates Mtb infection outcomes., (Copyright © 2020 Chandra et al.)

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) modulate differentiation and immune functions of macrophages (MΦ). Our aim was to evaluate how different MΦ differentiation conditions influence the MΦ response to virus infection. To address this, we differentiated bone marrow-derived MΦ in either GM-CSF or M-CSF and measured the cytokine responses to two different strains of lymphocytic choriomeningitis virus (LCMV) (clone 13; Cl13 or Armstrong; ARM). GM-CSF MΦ infected with either LCMV-ARM or -Cl13 produced more IL-6 than M-CSF MΦ, whereas M-CSF MΦ generated more IL-10 than GM-CSF MΦ. Interestingly, in M-CSF MΦ, LCMV-ARM induced more IL-10 production than Cl13. However, we could not detect any IL-12p70 or IL-23 after infection from either cell types. We also observed that GM-CSF MΦ was more efficient than M-CSF MΦ in supporting antigen-specific CD8 + T cell proliferation. Taken together, our data demonstrate that GM-CSF and M-CSF MΦ differ in how they respond to viral infection by their production of different cytokines, and their support for CD8 + T cell proliferation.

Bacillus Calmette‑Guérin (BCG) is considered to be a successful biotherapy for treating bladder cancer (BCa). However, the underlying mechanisms of BCG have not been completely clarified, to date. The role of macrophages in BCG therapy for BCa has still not been determined in vivo. In the present study, the role and potential mechanism of BCG (0.25, 1.25 and 6.25 µg/mouse; intravenous) immunotherapy for BCa was investigated in a NOD/scid IL2Rg‑/‑ (NSI) mouse model by targeting macrophages in vivo. Notably, it was observed that NSI mice with T24 BCa cells displayed high levels of the macrophage marker CD11b+ F4/80+ after injection via the tail vein of live BCG, as well as a significant reduction in tumor volume. The levels of the inflammatory and macrophage maturation cytokines, such as tumor necrosis factor‑α, interleukin (IL)‑1β, IL‑6, IL‑12P70, TNF superfamily member 11 and monocyte chemotactic protein 1, were significantly increased in the serum and the tumor supernatant compared to that in normal control subjects. Furthermore, BCG promoted the expression of the pro‑differential genes Spi‑1 proto‑oncogene, early growth response protein 1, nuclear factor (NF)‑κB and proto‑oncogene c‑Fos in bone marrow. In conclusion, these observations indicate that the injection of live BCG can target macrophages against bladder tumor growth in vivo. The mechanism is likely related to the promotion of macrophage maturation, immune activation and increased numbers of macrophages infiltrating the bladder tumor.

Although it has been reported that selenium (Se) deficiency can trigger inflammation, however, there are few reports on the effect of Se on the function of mouse peritoneal macrophages. Herein, we examined the expression of inflammatory factors, oxidative stress levels, and phagocytosis for primary-cultured peritoneal macrophages using control and Se-deficient groups. Our results revealed that Se deficiency induced the accumulation of oxygen free radicals and weakened antioxidant capacity. Se deficiency also significantly increased the expression of inflammation factors including iNOS, IL-1β, IL-12, IL-10, PTGe, and NF-κB. Meanwhile, Se suppression restrained macrophage production of TNF-α. The results of the phagocytosis assay demonstrated that Se deficiency inhibited the phagocytosis of macrophages. In conclusion, Se-deficient macrophages undergo severe inflammation through the NF-κB pathway due to the accumulation of oxygen free radicals and are hindered in their phagocytic capacity.

Lactoferrin (LF) has important bio-functions including immuno-modulation, while essential trace metals may interact with LF and thereby induce property especially bio-activity changes. Bovine LF was thus supplemented with Zn 2+ at 0.16, 0.32, and 0.64 mg/g LF to yield 10%, 20%, and 40% Zn-saturation, respectively. Afterwards, bovine LF and the Zn-supplemented LF products at 10-40-μg/mL doses were compared for their immuno-modulatory activities in two immune cells (murine splenocytes and RAW264.7 macrophages), using the stimulation index of the splenocytes, T lymphocyte subpopulations, macrophage phagocytosis, and cytokine production as evaluation reflectors. The results showed that bovine LF and the Zn-supplemented LF products had suppressive effect on the splenocytes and concanavalin A (ConA)- and lipopolysaccharide-stimulated splenocytes, but lower Zn-saturation and lower dose could alleviate and even counteract this suppressive effect (P < 0.05). More importantly, the Zn-supplemented LF product with lower Zn-saturation at lower dose exerted slightly higher macrophage stimulation, increased CD4 + /CD8 + ratio of T lymphocyte subpopulations, and were capable of enhancing the interleukin-2 (IL-2), IL-4, and interferon-γ production in the splenocytes or the IL-1β, IL-6, and tumor necrosis factor-α production in the macrophages significantly (P < 0.05). Contrary to its counterpart at lower dose, the Zn-supplemented LF product with higher Zn-saturation at higher dose mostly showed opposite effects in the two cell models. It is concluded that Zn supplementation has an impact on the immuno-modulation of bovine LF, while Zn-saturation is a key factor to modulate these assessed immune activities.

Macrophages are the preferential cell types to study various aspects of mycobacterial infection. Commonly used infection models for in-vitro studies are primary macrophages such as human monocyte derived macrophages (hMDMs) and macrophage like cell lines (THP-1). It is not clear if commercially available THP-1 cells can be used as hMDMs alternative for in-vitro M.tb infection experiments. We conducted a detailed investigation of the hMDM and THP-1 response to mycobacterial infection on a comparative basis and assess the most crucial aspects of infection which are most commonly studied. We assessed mycobacterial uptake and intracellular growth over time of a pathogenic drug-resistant and drug-susceptible M.tb strains (R179 and H37Rv) through colony forming units (CFUs). Both strains depicted similar uptake and intracellular growth in hMDMs and THP-1 macrophages over time (R179, p = 0.954) (H37Rv, p = 0.922). Cytotoxicity assays revealed a consistent viability up to day 16 post-infection across the strains in both THP-1 and hMDMs (R179, p = 0.271) (H37Rv, p = 0.068). Interestingly, both cell lines showed similar mycobacterial uptake and cellular viability in both susceptible as well as resistant M.tb strains. Cytokine/chemokine mRNA analysis through qPCR found no difference between cell types. Further, cytokine secretion measured through Luminex revealed no difference across the strains. Also, cytokine secretion analysis showed no difference in both cell lines across strains. In conclusion, our study shows that THP-1 and hMDMs bacterial uptake, viability and host response to drug-susceptible and drug-resistant mycobacterial infections are similar. Therefore, present study demonstrate that THP-1 cells are suitable substitutes for hMDMs for in-vitro M.tb infection experiments., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nutritional zinc deficiency leads to immune dysfunction and aggravates inflammation. However, the underlying mechanism remains unknown. In this study, the relationship between macrophage subtypes (M1 and M2) and helper T lymphocytes (Th1 and Th2) was investigated using the spleen from rats fed zinc-deficient or standard diet. In experiment I, 5-week-old male Sprague-Dawley rats were fed a zinc-deficient diet (without zinc additives) or a standard diet (containing 0·01% zinc) for 6 weeks. In experiment II, the rats were divided into four groups: one group was fed a standard diet for 6 weeks; two groups were fed zinc-deficient diets and were injected three times a week with either saline or interleukin-4 (IL-4) (zinc-deficient/IL-4 i.p.); a fourth group (zinc-deficient/standard) was fed a zinc-deficient diet for 6 weeks followed by a standard diet for 4 weeks. In experiment I; GATA-binding protein 3 (GATA-3) protein level, M2 macrophage, CD3 +  CD8 + cells, and IL-4/IL-13-positive cells significantly decreased in the spleens of the zinc-deficient group. Additionally, IL-1β and macrophage inflammatory protein-1α (MIP-1α) mRNA levels significantly increased in the splenic macrophages of the zinc-deficient group. In experiment II; M2 macrophages, CD3 +  CD8 + cells, IL-4/IL-13-positive cells, and GATA-3 protein levels significantly increased in the spleens of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Furthermore, IL-1β and MIP-1α mRNA levels decreased in the splenic macrophages of the zinc-deficient/IL-4 i.p. and zinc-deficient/standard groups. Zinc deficiency-induced aggravated inflammation is related to Th2 lymphocytes and followed by the association with loss of GATA-3, IL-4 and anti-inflammatory M2 macrophages. Importantly, IL-4 injection or zinc supplementation can reverse the effects of zinc deficiency on immune function., (© 2018 John Wiley & Sons Ltd.)

Trypanosoma cruzi, the causal agent of chronic Chagas cardiomyopathy, exhibits an important tropism for cardiac tissue. In consequence, T. cruzi experimental infection represents a unique model to study cardiac macrophage behavior and effector functions during either acute or chronic immune response. In this chapter we describe a protocol to isolate immune cells from T. cruzi-infected murine cardiac tissue and to determine the percentage, absolute number, phenotype, and functionality of monocytes and macrophages by using flow cytometry. Moreover, we describe the parameters to discriminate between resident and infiltrating mononuclear phagocytic cells within infected hearts. The investigations in this field will provide mechanistic insights about the roles of these innate immune cells in the context of a clinically relevant target tissue.

Brucella abortus can survive and replicate within host macrophages, and great efforts have been made to demonstrate the genes involved in pathogenicity, such as internalization, in Brucella research. Here, intracellular responses were compared between THP-1 macrophage cells stimulated with B. abortus wild-type and four mutants (C1, C10, C27, and C32) using microarray to demonstrate the role of genes related to intracellular survival and replication. These mutants were generated by deleting genes encoding BAB&#95;RS13225 (4-hydrobenzoate 3-monooxygenase, PHBH), BAB&#95;RS00455 (heme exporter protein cytochrome C, CcmC), BAB&#95;RS03675 (exopolyphosphatase, PPX), and BAB&#95;RS13225 (peptidase M24). The results showed that mutants C1 and C10 induced significant suppression of survival levels and cytokine expression relative to wild-type in the THP-1 macrophage cells. These findings suggest that the BAB&#95;RS13225 and BAB&#95;RS00455 genes play important roles in survival within human macrophages. Conversely, mutants C27 and C32 induced significantly higher survival level than wild-type in the cells inhibiting cellular signal transduction. It is assumed that the BAB&#95;RS03675 and BAB&#95;RS13225 genes play a role in cellular resistance to B. abortus . Therefore, the disrupted genes are involved in B. abortus intracellular growth, and especially in its survival, and they could be effective targets for understanding the intracellular bacterium, B. abortus .

Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages.

Tuberculosis, a human infectious disease caused by Mycobacterium tuberculosis ( M.tb ), is still a major cause of morbidity and mortality worldwide. The success of M.tb as a pathogen relies mainly on its ability to divert the host innate immune responses. One way by which M.tb maintains a persistent infection in a "silent" granuloma is to inhibit inflammation and induce an immunoregulatory phenotype in host macrophages (MΦs). However, M.tb effectors governing the switch of MΦs from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype remain to be determined. The E arly S ecreted A ntigenic T arget 6 kDa or ESAT-6, has been implicated in the virulence and pathogenesis of tuberculosis. Here, we investigated roles of ESAT-6 in MΦ differentiation and polarization. We found that treatment of human monocytes with ESAT-6 did not interfere with differentiation of M1 MΦs. However, ESAT-6 promoted differentiation of M0 and M2 MΦs toward the M1 phenotype, as indicated by secretion of pro-inflammatory cytokines IL-6, IL-12, and TNF-α, and induction of a typical M1 transcriptional signature. Interestingly, we found that ESAT-6 switched terminal full activation of M1 polarized MΦs to the M2 phenotype. Indeed, in the pro-inflammatory M1 MΦs, ESAT-6 was able to inhibit IL-12 and TNF-α secretion and stimulate that of IL-10. Moreover, gene expression profiling of these cells showed that ESAT-6 induced downregulation of M1 MΦ cell surface molecules CD80 and CD86 , transcription factors IRF5 and c-MAF , cytokines IL-12, IL-10 , and IL-6 , as well as chemokines CXCL10 and CXCL1 . Overall, our findings suggest ESAT-6 as being one of the effectors used by M.tb to induce the pro-inflammatory M1 phenotype at the primo-infection; a prerequisite step to promote granuloma formation and subsequently drive the phenotype switch of MΦ polarization from M1 to M2 at a later stage of the infection. Our study improves current knowledge regarding mechanisms of virulence of M.tb and may be helpful to develop novel tools targeting ESAT-6 for a better and more efficient treatment of tuberculosis.

Chronic inflammation is a potential systemic risk factor for many bladder dysfunctions, including interstitial cystitis (IC). However, the underlying mechanism through which a healthy bladder protects itself from inflammatory triggers remains unknown. In this study, we identified odor compounds in urine obtained from IC patients and healthy controls. Using comprehensive solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (SPME-GC-TOF-MS) profiling and bioinformatics, we found that levels of urinary volatile metabolites, such as menthol, were significantly reduced in IC patients, compared to healthy controls. In an attempt to understand the mechanistic meaning of our volatile metabolites data and the role of menthol in the immune system, we performed two independent experiments: (a) cytokine profiling, and (b) DNA microarray. Our findings suggest that lipopolysaccharide (LPS)-stimulated inflammatory events, such as the production and secretion of inflammatory cytokines (e.g., TNF-α, IL-6, and IL-1β) and the activation of NF-κB and associated proteins within a large signaling network (e.g., Akt, TLR1, TNFAIP3, and NF-κB), are suppressed by the presence of menthol. These findings broaden our knowledge on the role of urinary menthol in suppressing inflammatory events and provide potential new strategies for alleviating both the odor and inflammation associated with IC.

Background: The delivery of plasmonic particles, such as gold nanorods, to the tumor microenvironment has attracted much interest in biomedical optics for topical applications as the photoacoustic imaging and photothermal ablation of cancer. However, the systemic injection of free particles still crashes into a complexity of biological barriers, such as the reticuloendothelial system, that prevent their efficient biodistribution. In this context, the notion to exploit the inherent features of tumor-tropic cells for the creation of a Trojan horse is emerging as a plausible alternative., Results: We report on a convenient approach to load cationic gold nanorods into murine macrophages that exhibit chemotactic sensitivity to track gradients of inflammatory stimuli. In particular, we compare a new model of poly-L-lysine-coated particles against two alternatives of cationic moieties that we have presented elsewhere, i.e. a small quaternary ammonium compound and an arginine-rich cell-penetrating peptide. Murine macrophages that are exposed to poly-L-lysine-coated gold nanorods at a dosage of 400 µM Au for 24 h undertake efficient uptake, i.e. around 3 pg Au per cell, retain the majority of their cargo until 24 h post-treatment and maintain around 90% of their pristine viability, chemotactic and pro-inflammatory functions., Conclusions: With respect to previous models of cationic coatings, poly-L-lysine is a competitive solution for the preparation of biological vehicles of gold nanorods, especially for applications that may require longer life span of the Trojan horse, say in the order of 24 h. This biopolymer combines the cost-effectiveness of small molecules and biocompatibility and efficiency of natural peptides and thus holds potential for translational developments.

Background: Type 2 diabetes (T2D) is a risk factor for the development of tuberculosis (TB), although the associated mechanisms are not known., Objectives: To study the association between T2D and the basal phenotype of macrophages, and their immune response to Mycobacterium tuberculosis (Mtb) infection., Methods: We evaluated the influence of T2D on the response of monocyte-derived macrophages (MDM) to Mtb in patients with T2D (n = 10) compared to healthy subjects (n = 9), before and after infection with Mtb clinical isolates bearing different degrees of virulence. The levels of cell surface markers for activation secreted cytokines and chemokines, bacterial association, and intracellular bacterial growth were evaluated., Findings: The expression levels of HLA-DR, CD80, and CD86 were low while those of of PD-L1 were high in uninfected MDMs derived from patients with diabetes; as a result of Mtb infection, changes were only observed in the expression levels of PD-L1. The levels of cytokines (e.g., IL-6, IL-1β, IL-10, and IL-12) and chemokines (e.g., MCP-1, MIG, and RANTES) are perturbed in MDMs derived from patients with diabetes, both before infection and in response to Mtb infection. In response to the more virulent Mtb strains, the levels of association and bacterial clearance were diminished in MDMs derived from patients with diabetes., Conclusions: T2D affects the basal activation state of the macrophages and its capacity to respond and control Mtb infection.

Background: Guang-Pheretima, which is originated from Pheretima aspergillum, has been documented in academic Chinese herbal studies for nearly 2000 years for its prominent treating effects of various inflammatory diseases such as asthma, cough and fever. However, the anti-inflammatory activity and mechanism of Guang-Pheretima has been rarely reported. Hence, we investigated the inhibitory effect and the underlying mechanism of Guang-Pheretima aqueous extracts on inflammatory response in RAW 264.7 cells., Method: RAW 264.7 macrophages were pretreated with various concentrations of Guang-Pheretima decoction (GPD) or protein-free Guang-Pheretima decoction (PF-GPD) and subsequently stimulated with lipopolysaccharide (LPS) to trigger the inflammatory response. Productions of nitric oxide (NO) were determined by Griess reaction, and prostaglandin E 2 (PGE 2 ), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 were measured by enzyme-linked immunosorbent assays (ELISA). The protein expressions and messenger ribonucleic acid (mRNA) amounts of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were analyzed by Western Blot and Real-Time polymerase chain reaction (PCR), respectively. Finally, the translocation of nuclear factor (NF)-κB was observed by Western Blot., Results: GPD of the experimental concentrations showed no anti-inflammatory activity. In contrast, PF-GPD at concentrations of 40-320 μg/mL significantly inhibited NF-κB activation and reduced the production of inflammatory mediators, such as NO, PGE 2 , TNF-α, as well as the related key synthases including iNOS and COX-2. Moreover, PF-GPD markedly suppressed the release of inflammatory cytokines, such as IL-1β and IL-6., Conclusion: These results demonstrate the excellent anti-inflammatory properties of PF-GPD, and suggest that Guang-Pheretima may be used to treat and prevent certain inflammatory diseases.

The tropical bont tick, Amblyomma variegatum , is a tick species of veterinary importance and is considered as one of major pest of ruminants in Africa and in the Caribbean. It causes direct skin lesions, transmits heartwater, and reactivates bovine dermatophilosis. Tick saliva is reported to affect overall host responses through immunomodulatory and anti-inflammatory molecules, among other bioactive molecules. The general objective of this study was to better understand the role of saliva in interaction between the Amblyomma tick and the host using cellular biology approaches and proteomics, and to discuss its impact on disease transmission and/or activation. We first focused on the immuno-modulating effects of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages in vitro . We analyzed its immuno-suppressive properties by measuring the effect of saliva on PBMC proliferation, and observed a significant decrease in ConA-stimulated PBMC lymphoproliferation. We then studied the effect of saliva on bovine macrophages using flow cytometry to analyze the expression of MHC-II and co-stimulation molecules (CD40, CD80, and CD86) and by measuring the production of nitric oxide (NO) and pro- or anti-inflammatory cytokines. We observed a significant decrease in the expression of MHC-II, CD40, and CD80 molecules, associated with decreased levels of IL-12-p40 and TNF-α and increased level of IL-10, which could explain the saliva-induced modulation of NO. To elucidate these immunomodulatory effects, crude saliva proteins were analyzed using proteomics with an Orbitrap Elite mass spectrometer. Among the 336 proteins identified in A. variegatum saliva, we evidenced bioactive molecules exhibiting anti-inflammatory, immuno-modulatory, and anti-oxidant properties (e.g., serpins, phospholipases A2, heme lipoprotein). We also characterized an intriguing ubiquitination complex that could be involved in saliva-induced immune modulation of the host. We propose a model for the interaction between A. variegatum saliva and host immune cells that could have an effect during tick feeding by favoring pathogen dissemination or activation by reducing the efficiency of host immune response to the corresponding tick-borne diseases.

Macrophages are among the main cells involved in generation of inflammation. To investigate the anti-inflammatory effect of Satureja hortensis (summer savory), lipopolysaccharide (LPS)-activated J774.1 macrophages were treated with various extracts, and the expression and release of various inflammatory molecules by macrophages were examined. We showed that dichloromethane and hexane extracts reduced nitric oxide (NO) production more efficiently than other extracts. Both extracts decreased gene expression of inducible NO synthase (iNOS) (<0.44 fold of control), cyclooxygenase (COX)-2 (<0.29 fold), interleukin (IL)-1β (<0.41 fold), IL-6 (<0.25 fold) and tumor necrosis factor (TNF)-α (<0.2 fold). The extracts reduced IL-6 and IL-1β proteins production from macrophages. Surface intensity of expression of intercellular adhesion molecule (ICAM)-1 decreased to 845 ± 28.1 (dichloromethane) and 715 ± 48.6 (hexane) compared to the control (902 ± 73.1). These findings showed that Satureja hortensis, by influencing macrophages and related mediators, could contribute to reduction of inflammation and might be useful as an anti-inflammatory agent.

Background: Cigarette smoking increases systemic oxidative stress, inflammation, and viral replication in individuals with HIV. Macrophages are infected during HIV infection and serve as an important reservoir throughout the process. Macrophages exist in two phenotypes, the classically activated M1 macrophage and alternatively activated M2 macrophage. The expression of drug efflux transporters and metabolic enzymes, which have direct effects on intracellular drug concentrations, differ between the pro-inflammatory M1 macrophage and the anti-inflammatory M2 macrophage., Objective: To further explain the role of tobacco use in worsened outcomes in the HIV + population receiving antiretroviral therapy., Methods: Western blotting was used to examine macrophage polarization and expression of drug efflux transporters, CYP enzymes, and antioxidant enzymes. The arginase assay was used to measure arginase activity. Cytokine production was measured using the human multiplex inflammatory cytokine assay kit. The 8-OHdG DNA Damage Quantification Direct Kit was used to quantify DNA damage. Viral replication under the influence of tobacco and antiretroviral drug use was measured by p24 Elisa., Results: We observed phenotypic shifts from M1 to M2 with both individual and combination treatments with cigarette smoke condensate and the protease inhibitor antiretroviral drug lopinavir. These shifts lead to changes in cytokine production, the expression of CYP enzymes, anti-oxidant enzymes, and drug efflux transporters, as well as changes in viral replication., Conclusion: This data suggest a mechanism by which tobacco use impairs HIV antiretroviral therapy to increase intracellular drug concentrations in this important cellular reservoir., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The present study aimed to investigate the effects of polysaccharides extracted from Bupleurum chinense DC (BCPs) on macrophage functions. In the in vivo experiment, 1 mL of 5% sodium thioglycollate was injected into the abdomen of the mice on Day 0 and macrophages were harvested on Day 4. The macrophages were cultured in plates and treated with different concentrations of BCPs and stimulus. Effects of BCPs on macrophage functions were assessed by chemotaxis assay, phagocytosis assay and Enzyme-Linked Immunosorbent Assay (ELISA). Our results showed the enhanced chemotaxis, phagocytosis and secretion of nitric oxide (NO) and inflammatory cytokines by macrophages when treated with BCPs. However, when chemotaxis and phagocytosis were up-regulated by complement components or opsonized particles, BCPs inhibited these effects. Also, the NO production induced by lipopolysaccharides (LPS) was suppressed by BCPs mildly. Moreover, BCPs had an inhibitory effect on the [Ca 2+ ] i elevation of macrophages. These results suggested that BCPs exerted modulatory effects on macrophage functions, which may contribute to developing novel approaches to treating inflammatory diseases., (Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Background: During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa., Results: Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria., Conclusions: Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

In this study, the egg yolk livetins (α, β, and γ-livetin) fraction and its hydrolysates, prepared by pepsin and Alcalase, were evaluated for their anti-inflammatory effects using lipopolysaccharide (LPS)-induced RAW 264.7 macrophages as an in vitro model. Enzymatic hydrolysis by pepsin and Alcalase successfully transformed the large molecular weight livetins into low molecular mass peptides mostly below 10kDa. Results revealed that livetins and its hydrolysates (peptides) treatment significantly reduced the inflammatory responses as evidenced by inhibition of production of nitric oxide (NO) (22.7-39.2%), pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) (36.9-43.2%), interleukin-1β (IL-1β) (26.1-50.9%) and interleukin-6 (IL-6) (60.4-69.0%), and the expression of inducible nitric oxide synthase (iNOS) (58.6-62%). Alcalase hydrolysate showed more effects in inhibiting prostaglandin-E2 (PGE2) production (30.3%) as well as expression of cyclooxygenase-2 (COX-2) (55.7%). In addition, effect of livetins and its hydrolysates on phagocytic capacity of the macrophages was also evaluated. The results indicate that livetins and its enzymatic hydrolysates significantly (p<0.001, 0.05) enhanced the phagocytic activity of the macrophages. The results suggest that egg yolk livetins and its hydrolysates with anti-inflammatory activity can potentially be used in health food/nutraceutical/pharmaceutical industry for various applications., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Macrophages are involved in all phases of scaffold induced tissue regeneration, orchestrating the transition from an inflammatory to regenerative phenotype to guide all other cell types to complete the wound healing process when a tissue defect advances beyond the critical size. Therefore, harnessing macrophages by scaffolds is important for facilitating tissue regeneration in situ. In this work we utilized the superparamagnetic scaffold upon magnetization as a mechanostimulation platform to apply forces directly to macrophages grown in the scaffold, aiming to figure out whether the functions of macrophages related to bone tissue regeneration can be mechanomodulated and to elucidate the underlying mechanisms. We showed the first evidence that upon magnetization the interaction of superparamagnetic scaffolds to macrophages drove them to polarize towards an M2-like phenotype by inhibiting TLR2/4 activation and enhancing VEGFR2 activation, thereby inhibiting secretion of the pro-inflammatory cytokines IL-1β, TNF-α and MCP-1, as well as the osteoclast differentiation cytokines MMP-9 and TRAP, and up-regulating VEGF and PDGF. The conditioned media enhanced the osteogenesis of osteoblasts and the angiogenesis of endothelial cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To investigate whether short-term, systemic depletion of macrophages can mitigate osteoarthritis (OA) following injury in the setting of obesity., Methods: CSF-1R-GFP+ macrophage Fas-induced apoptosis (MaFIA)-transgenic mice that allow conditional depletion of macrophages were placed on a high-fat diet and underwent surgery to induce knee OA. A small molecule (AP20187) was administrated to deplete macrophages in MaFIA mice. The effects of macrophage depletion on acute joint inflammation, OA severity, and arthritic bone changes were evaluated using histology and micro-computed tomography. Immunohistochemical analysis was performed to identify various immune cells. The levels of serum and synovial fluid cytokines were also measured., Results: Macrophage-depleted mice had significantly fewer M1 and M2 macrophages in the surgically operated joints relative to controls and exhibited decreased osteophyte formation immediately following depletion. Surprisingly, macrophage depletion did not attenuate the severity of OA in obese mice; instead, it induced systemic inflammation and led to a massive infiltration of CD3+ T cells and particularly neutrophils, but not B cells, into the injured joints. Macrophage-depleted mice also demonstrated a markedly increased number of proinflammatory cytokines including granulocyte colony-stimulating factor, interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor in both serum and joint synovial fluid, although the mice showed a trend toward decreased levels of insulin and leptin in serum after macrophage depletion., Conclusion: Our findings indicate that macrophages are vital for modulating homeostasis of immune cells in the setting of obesity and suggest that more targeted approaches of depleting specific macrophage subtypes may be necessary to mitigate inflammation and OA in the setting of obesity., (© 2017, American College of Rheumatology.)

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO 3 )-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO 3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO 3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO 3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO 3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO 3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO 3 particles., (Copyright © 2017 Elsevier Inc. All rights reserved.)

As an important structural analogue of bisphenol A (BPA), bisphenol S (BPS) has been used as alternatives to BPA in industrialized production. However, the immunotoxicity of BPS remains poorly understood. As a critical model in inflammatory responses, macrophages are used to explore the immunotoxic potential and mechanisms of BPS at environmentally relevant concentrations in our study. Here, we are combining molecular toxicology and mass spectrometry (MS)-based global metabolomics and lipidomics study together to estimate the variation of cytokines profiling and metabolism characteristic following BPS exposure. Our results demonstrated that BPS exposure induced pro-inflammatory phenotype by activating the immuno-related cytokines which include TNF-α, IL-1β and IL-6, modulating metabolic pathways which include glycolytic, glutathione (GSH), sphingomyelin (SM)-ceramide (Cer), glycerophospholipids (GPs) and glycerolipids (GLs). These toxicological mechanisms are providing us with a deeper understanding of the critical role of metabolites and lipids reprogramming in immunotoxicity of BPS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, endothelial-mesenchymal transition (EndMT) has been demonstrated to play an important role in the development of atherosclerosis, the molecular mechanisms of which remain unclear. In the present study, scanning electron microscopy directly revealed a widened endothelial space and immunohistofluorescence demonstrated that EndMT was increased in human aorta atherosclerotic plaques. M1 macrophage-derived foam cell (M1-FC) supernatants, but not M2 macrophage-derived foam cell (M2-FC) supernatants, induced EndMT. A protein array and enzyme-linked immunosorbent assay identified that the levels of several cytokines, including C-C motif chemokine ligand 4 (CCL-4) were increased in M1-FC supernatants, in which EndMT was promoted, accompanied by increased endothelial permeability and monocyte adhesion. Furthermore, anti-CCL-4 antibody abolished the effects of M1-FC supernatants on EndMT. At the same time, CCL-4 activated its receptor, C-C motif chemokine receptor-5 (CCR-5), and upregulated transforming growth factor-β (TGF-β) expression. Further experiments revealed that EndMT induced by CCL-4 was reversed by treatment with CCR-5 antagonist and the RNA-mediated knockdown of TGF-β. On the whole, the data of the present study suggest that M1-FCs induce EndMT by upregulating CCL-4, and increase endothelial permeability and monocyte adhesion. These data may help to elucidate the important role of EndMT in the development of atherosclerosis.

The progression of human immunodeficiency virus (HIV) is associated with mucosal damage in the gastrointestinal (GI) tract. This damage enables bacterial translocation from the gut and leads to subsequent inflammation. Dextran sulfate sodium (DSS-exposure) is an established animal model for experimental colitis that was recently shown to recapitulate the link between GI-tract damage and pathogenic features of SIV infection. The current study tested the protective properties of ABX464, a first-in-class anti-HIV drug candidate currently in phase II clinical trials. ABX464 treatment strongly attenuated DSS-induced colitis in mice and produced a long-term protection against prolonged DSS-exposure after drug cessation. Consistently, ABX464 reduced the colonic production of the inflammatory cytokines IL-6 and TNFα as well as that of the chemoattractant MCP-1. However, RNA profiling analysis revealed the capacity of ABX464 to induce the expression of IL-22, a cytokine involved in colitis tissue repair, both in DSS-treated mice and in LPS-stimulated bone marrow-derived macrophages. Importantly, anti-IL-22 antibodies significantly reduced the protective effect of ABX464 on colitis in DSS-treated mice. Because reduced IL-22 production in the gut mucosa is an established factor of HIV and DSS-induced immunopathogenesis, our data suggest that the anti-inflammatory properties of ABX464 warrant exploration in both HIV and inflammatory ulcerative colitis (UC) disease.

Chronic non-communicable diseases such as obesity are preceded by increased macrophage infiltration in adipose tissue and greater secretion of pro-inflammatory cytokines. We evaluated the anti-inflammatory potential of Biotransformed extract, and two control extracts: In Natura and Autoclaved. The assays were performed using a cellular model with RAW264.7, 3T3-L1 cells, and RAW264.7 and 3T3-L1 co-culture. The innovation of the study was the use of Biotransformed extract, a unique phenolic extract of a bioprocessed citrus residue. LPS stimulated RAW264.7 cells treated with the Biotransformed extract exhibited lower secretion of TNF-α and NO and lower protein expression of NFκB. In RAW264.7 and 3T3-L1 co-culture, treatment with 1.0mg/mL of the Biotransformed extract reduced secretion of TNF-α (30.7%) and IL-6 (43.4%). Still, the Biotransformed extract caused higher increase in adiponectin in relation to control extracts. When the co-culture received a LPS stimulus, the Autoclaved extract at 1.0mg/mL reduced IL-6 and TNF-α concentrations, and raised adiponectin. However, it was noteworthy that the Biotransformed extract was also able to significantly reduce IL-6 concentration while the Natural extract was not. The Biotransformed citrus extract evaluated in this study showed anti-inflammatory activity in macrophages and in co-culture, indicating that bioprocess of citrus residue can contribute to new product development with anti-inflammatory potential., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To investigate the role of human umbilical cord mesenchymal stem cells (hucMSCs) in the treatment of dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD)., Results: ICG-hucMSCs homed to colon tissues of IBD mice 12 h after injection. The injection of hucMSCs significantly relieved the IBD symptoms and inflammatory cell infiltration. The expression of IL-10 gene increased while those of 15-LOX-1, TNF-α, IL-6, IL-1β, and IP-10 genes decreased in colon tissues and spleens of hucMSCs-treated mice. The activation of STAT3 was inhibited in colon tissues and spleens of IBD mice that were treated with hucMSCs. In addition, the percentage of macrophages decreased in colon tissues and spleens of hucMSCs-treated IBD mice. Moreover, we provided evidence that in vitro co-culture with hucMSCs inhibited the expression of 15-LOX-1, IL-6 and p-STAT3 in mouse enterocoelia macrophages., Conclusions: HucMSCs alleviate DSS-induced IBD through the modulation of 15-LOX-1 in macrophages.

Lithium (Li) is a chemical element used for treating and preventing bipolar disorder (BD) and exerts positive effects such as anti-inflammatory effects as well as undesirable side effects. These effects of Li can be influenced by interaction with some nutritional elements. Therefore, we investigated the potential effects of xanthine (caffeine and theobromine) and catechin molecules present in some food beverages broadly consumed worldwide, such as coffee and tea, on Li-induced anti-inflammatory effects. In the present study, we concomitantly exposed RAW 264.7 macrophages to Li, isolated xanthine and catechin molecules, and a xanthine-catechin mixture (XC mixture). We evaluated the effects of these treatments on cell proliferation, cell cycle progression, oxidative and antioxidant marker expression, cytokine levels, gene expression, and GSK-3 β enzyme expression. Treatment with the XC mixture potentialized Li-induced anti-inflammatory effects by intensification of the following: GSK-3 β inhibitory action, lowering effect on proinflammatory cytokines (IL-1 β , IL-6, and TNF α ), and increase in the levels of IL-10 that is an anti-inflammatory cytokine. Despite the controversial nature of caffeine consumption by BD patients, these results suggested that consumption of caffeine, in low concentrations, mixed with other bioactive molecules along with Li may be safe.

In this study, we evaluated structure characterization and immunomodulatory activity of polysaccharides from Astragalus aboriginum Richardson (RAPS), Atractylodes macrocephala Koidz (RAMPS) and Rumia seseloides Hoffm (RSPS) in vitro on chicken macrophage. We found that molecular weight of RAPS and RAMPS was 122.4 and 109.4kDa higher than 64.71kDa of RSPS. Glucose occupied 83.95% and 66.39% in RAPS and RAMPS, respectively. RSPS mainly contained glucose and galacturonic acid, which accounted for 32.35% and 29.25%, respectively. The NMR results displayed that RAPS and RAMPS contained β- glucose, β-galactose, and β-galacturonic acid. The backbone was 1→6 linked glucose. RSPS showed at least six monosaccharide response signals. In vitro experiment, the results showed that RAPS at dosage of 15.62μgmL(-1) exhibited significant immunological on chicken macrophage compared to RAMPS and RSPS. Interestingly, costimulatory molecules levels in RSPS group were higher than that of RAPS, which may associated with the special structure of RSPS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The host immune response to bone biomaterials is vital in determining the fate of scaffolds and also the outcomes of bone regeneration. Mineralized collagen is an ideal tissue-engineering scaffold for bone repair; however, little is known about its immunomodulatory properties after implantation. In this study, extrafibrillarly-mineralized collagen (EMC) and intrafibrillarly-mineralized collagen (IMC) scaffolds with different nanostructures were fabricated and their immunomodulatory properties via macrophage polarization during bone regeneration were investigated. Micro-CT findings showed that the IMC scaffold yielded more new bone formation than the EMC scaffold. In the defect area, more CD68 + CD163 + M2-like macrophages were observed in the IMC group, while M1-like macrophages positive for CD68 and inducible nitric oxide synthase (iNOS) increased dramatically in the EMC group. We further demonstrated, from the protein and RNA levels, that M2-associated anti-inflammatory cytokines interleukin (IL)-10 and arginase-1 were highly expressed in the macrophages seeded on the IMC scaffold, while those seeded on the EMC scaffold expressed more M1-related genes iNOS and IL-6. Moreover, the macrophage polarization in response to the nanostructure of mineralized collagen scaffolds influenced the osteogenesis of human bone marrow stromal cells. These findings suggest that the nanostructure of mineralized collagen scaffolds affects macrophage functional polarization during bone regeneration. The immunomodulatory properties of biomaterial scaffolds can be a dictator of bone regeneration outcomes.

The extracellular matrix molecules remaining in bioscaffolds derived from decellularized xenogeneic tissues appear to be important for inducing cell functions conducting tissue regeneration. Here, we studied whether decellularization methods, that is, detergent Triton X-100 (TX) alone and TX combined with reversible alkaline swelling (STX), applied to bovine pericardial tissue, could affect the bioscaffold components. The in vitro macrophage response, subdermal biodegradation, and cell infiltration were also studied. The results indicate a lower leaching of fibronectin, but a higher leaching of laminin and sulfated glycosaminoglycans from tissues decellularized with STX and TX, respectively. The in vitro secretion of interleukin-6 and monocyte chemoattractant protein by RAW264.7 macrophages is promoted by decellularized bioscaffold leachates. A lower polymorphonuclear cell density is observed around decellularized bioscaffolds at 1-day implantation; concurrently showing a higher cell infiltration in STX- than in TX-implant. Cells infiltrated into TX-implant show a fibroblastic morphology at 7-day implantation, concurrently the capillary formation is observed at 14-day. Pericardial bioscaffolds suffer biodegradation more pronounced in STX- than in TX-implant. Both TX and STX decellularization methods favor a high leaching of basal lamina components, which presumably promotes a faster macrophage stimulation compared to nondecellularized tissue, and appear to be associated with an increased host cell infiltration in a rat subdermal implantation. Meanwhile, the connective tissue components leaching from TX decellularized bioscaffolds, unlike the STX ones, appear to be associated with an enhanced angiogenesis accompanied by an early-promoted fibroblastic cell transition. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2810-2822, 2016., (© 2016 Wiley Periodicals, Inc.)

Macrophages constitute the first line of defense against Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development., Competing Interests: The authors have declared that no competing interests exist.

Macrophage activation and persistent inflammation contribute to the pathogenesis of spinal cord injury (SCI), and different phenotypes of macrophages play diverse roles in the pathological process of SCI. After SCI, there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound. The long-lasting predominance of M1 macrophages may derail healing and compromise organ functions. Based on the previous findings that the transcription factor interferon regulatory factor 5 (IRF5) up-regulates genes associated with M1 macrophages, we attempted to examine the effect of silencing IRF5 on SCI progression. IRF5 expression was assessed with Western blotting or immunohistochemistry. Macrophage phenotypes were measured with flow cytometry or immunohistochemistry. M1- or M2-related cytokines were measured with a Luminex assay kit. IRF5 siRNA was delivered into the macrophages infiltrated into the wound of SCI mice through lipidoid nanoparticle. Locomotor functions were measured with Basso Mouse Scale (BMS) scoring. Myelination was assessed with luxol fast blue staining. Myelin binding protein, neurofilaments, synaptic markers, and cytokines in the wound area were measured with Western blotting. The Mann-Whitney U test was used for statistical analyses. After SCI, significant elevation of IRF5 was evident on day 1, peaked on day 7, and gradually decreased thereafter. Similar dynamic change in the expression of CD86, a typical M1 marker, was observed. In contrast, there was a transient increase in the expression of CD206, a typical M2 marker, which peaked 6 h after SCI, and returned to baseline within 1 day. Macrophages isolated from the epicenter at day 3 after SCI were predominantly M1 phenotype, and a siRNA-mediated knockdown of IRF5 resulted in a reduced expression of M1 macrophage markers and increased expression of M2 macrophage markers. Nanoparticle-mediated delivery of IRF5 siRNA to SCI mouse model resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function (p < 0.05). IRF5 may serve as a therapeutic target to promote post-SCI recovery.