Expression Of Phagocytic Receptors Research Articles

Phenotypic and Functional Outcomes in Macrophages Exposed to an Environmentally Relevant Mixture of Organophosphate Esters in Vitro.

Organophosphate esters (OPEs) are flame retardants and plasticizers used in consumer products. OPEs are found ubiquitously throughout the environment with high concentrations in indoor house dust. Exposure to individual OPEs is associated with immune dysfunction, particularly in macrophages. However, OPEs exist as complex mixtures and the effects of environmentally relevant mixtures on the immune system have not been investigated. The objectives of this study were to evaluate the toxicity of an environmentally relevant mixture of OPEs that models Canadian house dust on macrophages using phenotypic and functional assessments in vitro. High-content live-cell fluorescent imaging for phenotypic biomarkers of toxicity in THP-1 macrophages treated with the OPE mixture was undertaken. We used confocal microscopy and cholesterol analysis to validate and expand on the observed OPE-induced lipid phenotype. Then, we used flow cytometry and live-cell imaging to conduct functional tests and uncover mechanisms of OPE-induced phagocytic suppression. Finally, we validated our THP-1 findings in human primary peripheral blood mononuclear cells (hPBMC) derived macrophages. Exposure to non-cytotoxic dilutions of the OPE mixture resulted in higher oxidative stress and disrupted lysosome and lipid homeostasis in THP-1 and primary macrophages. We further observed that phagocytosis of apoptotic cells in THP-1 and primary macrophages was lower in OPE-exposed cells vs. controls. In THP-1 macrophages, phagocytosis of both Gram-positive and Gram-negative bacteria was also lower in OPE-exposed cells vs. controls. Additionally, the OPE mixture altered the expression of phagocytic receptors linked to the recognition of phosphatidylserine and pathogen-associated molecular patterns. The results of this in vitro study suggested that exposure to an environmentally relevant mixture of OPEs resulted in higher lipid retention in macrophages and poor efferocytic response. These effects could translate to enhanced foam cell generation resulting in higher cardiovascular mortality. Furthermore, bacterial phagocytosis was lower in OPE-exposed macrophages in an in vitro setting, which may indicate the potential for reduced bacterial clearance in models of infections. Taken together, our data provide strong evidence that mixtures of OPEs can influence the biology of macrophages and offer new mechanistic insights into the impact of OPE mixtures on the immune system. https://doi.org/10.1289/EHP13869.

Mass cytometric analysis of the immune cell landscape after traumatic brain injury elucidates the role of complement and complement receptors in neurologic outcomes

Following traumatic brain injury (TBI), a neuroinflammatory response can persist for years and contribute to the development of chronic neurological manifestations. Complement plays a central role in post-TBI neuroinflammation, and C3 opsonins and the anaphylatoxins (C3a and C5a) have been implicated in promoting secondary injury. We used single cell mass cytometry to characterize the immune cell landscape of the brain at different time points after TBI. To specifically investigate how complement shapes the post-TBI immune cell landscape, we analyzed TBI brains in the context of CR2-Crry treatment, an inhibitor of C3 activation. We analyzed 13 immune cell types, including peripheral and brain resident cells, and assessed expression of various receptors. TBI modulated the expression of phagocytic and complement receptors on both brain resident and infiltrating peripheral immune cells, and distinct functional clusters were identified within same cell populations that emerge at different phases after TBI. In particular, a CD11c+ (CR4) microglia subpopulation continued to expand over 28 days after injury, and was the only receptor to show continuous increase over time. Complement inhibition affected the abundance of brain resident immune cells in the injured hemisphere and impacted the expression of functional receptors on infiltrating cells. A role for C5a has also been indicated in models of brain injury, and we found significant upregulation of C5aR1 on many immune cell types after TBI. However, we demonstrated experimentally that while C5aR1 is involved in the infiltration of peripheral immune cells into the brain after injury, it does not alone affect histological or behavioral outcomes. However, CR2-Crry improved post-TBI outcomes and reduced resident immune cell populations, as well as complement and phagocytic receptor expression, indicating that its neuroprotective effects are mediated upstream of C5a generation, likely via modulating C3 opsonization and complement receptor expression.

Capsular polysaccharide mediates Streptococcus agalactiae to resist Nile tilapia macrophage phagocytosis

Streptococcus agalactiae (S. agalactiae) is a highly pathogenic gram-positive bacterium for farmed tilapia. The surface capsular polysaccharide (cps) containing terminal sialic acid (sia) residues is a crucial virulence factor for S. agalactiae. However, there is limited knowledge regarding the effects of cps and sia on the phagocytosis of S. agalactiae by teleost macrophages (MФ). In this study, we investigated the role of cps and sia in tilapia MФ phagocytosis of S. agalactiae using cps-deficient mutant ∆cps and sia-deficient mutant ∆neuA. Nonopsonic phagocytosis of wild type (WT), ∆cps and ∆neuA was initially analyzed by flow cytometry. The results indicated that MФ phagocytosis of ∆cps and ∆neuA was significantly higher than that of WT. The proportion of MФ with higher intracellular reactive oxygen species (ROS) or nitric oxide (NO) levels, and overall mean fluorescence intensity (MFI) of the ROS or NO in MФ increased higher during nonopsonic phagocytosis in the Δcps and ΔneuA. The expression levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) during S. agalactiae infection revealed that S. agalactiae could dampen the inflammatory response of MФ through the cps and sia. Increased susceptibility of the ∆cps and ∆neuA to phagocytic killing indicated that S. agalactiae could impair the phagocytic clearance of MФ through the cps or sia. Furthermore, opsonic phagocytosis of the ∆cps and ∆neuA by MФ was dramatically enhanced compared to the WT under complement or antibody mediation. Moreover, relative to ∆cps and ∆neuA, nonopsonic and opsonic phagocytic receptors were significantly down-regulated during MФ phagocytosis of WT, indicating that S. agalactiae might resist phagocytosis of MФ by directly or indirectly inhibiting the expression of phagocytic receptors through the cps or sia. Our data suggest that cps plays a crucial role in the interference of S. agalactiae on the phagocytosis of tilapia MФ, and sia may be a significant immune escape site. These results provide valuable insights into the mechanism of S. agalactiae attempting to escape the defenses of teleost innate immune system and offer clues for S. agalactiae vaccine development.

A dual macrophage polarizer conjugate for synergistic melanoma therapy

Tumor associated macrophages (TAMs) play a paradoxical role in the fate of aggressive tumors like melanoma. Immune modulation of TAMs from the tumor-permissive M2 phenotype to antitumoral M1 phenotype is an emerging attractive approach in melanoma therapy. Resiquimod is a TLR7/8 agonist that shifts the polarization of macrophages towards M1 phenotype. Bexarotene (BEX) is a retinoid that induce the expression of phagocytic receptors in macrophages besides its ability to downregulate the M2 polarization. However, the clinical use of both agents is hindered by poor pharmacokinetic properties. Here, for the first time we repurposed BEX based on its immunomodulatory properties and combined it with RES by designing hyaluronic acid (HA) conjugates of both drugs that act synergistically as a dual macrophage polarizer to promote the M1 phenotype and suppress the M2 phenotype. This combination enhanced the macrophage secretion of proinflammatory cytokines (IL-6 and TNF-α), while suppressing the production of tumor promoting cytokine CCL22. It enhanced the macrophage phagocytic ability and showed superior inhibitory effects against B16F10 cells. In vivo studies on a mouse melanoma model confirmed the superiority of the dual conjugate compared to the single HA-drug conjugates in suppressing the tumor growth. Immunoprofiling of the excised tumors revealed a significant increase in the M1/M2 ratio of TAMs in mice treated with the dual conjugate. Our intravenously injectable HA conjugate of RES and BEX provides a promising immunotherapeutic combination strategy for resetting the M1/M2 ratio, supporting the tumoricidal activity of TAMs for effective melanoma treatment.

Decreased expression of a phagocytic receptor Siglec-1 on alveolar macrophages in chronic obstructive pulmonary disease

BackgroundAlveolar macrophages are professional phagocytes that remove microbial pathogens inhaled into the lung. The phagocytic ability is compromised in chronic obstructive pulmonary disease (COPD). However, the molecular mechanisms underlying this defect in phagocytosis are not clearly defined.Materials and methodsCell suspensions were collected from lung tissues of patients undergoing lung resection. Alveolar macrophages were detected as FSChi/ SSChi/CD45+/CD206+ cells in the isolated cell suspension by flow-cytometry. The cell surface expression of plasma membrane-bound phagocytic receptors (Fcγ receptor I (FcγRI), a complement receptor CD11b, macrophage scavenger receptor-1 (MSR-1), CD36 and Siglec-1) was determined on the alveolar macrophages. Correlations between the expression levels of the phagocytic receptors and disease severity were analysed. Phagocytosis of fluorescence-tagged bacteria by human alveolar macrophages was evaluated.ResultsThe flow-cytometry analyses revealed that FcγRI, CD11b, MSR-1 and Siglec-1, but not CD36, were expressed on human alveolar macrophages. Among these receptors, Siglec-1 expression was significantly decreased on alveolar macrophages in COPD ex-smokers (n = 11), compared to control never-smokers (n = 11) or control ex-smokers (n = 9). The Siglec-1 expression on alveolar macrophages was significantly correlated with lung function (forced expiratory volume in 1 s) and with the severity of emphysema. Treatment of human alveolar macrophages with an anti-Siglec1 blocking antibody decreased phagocytosis of non-typeable Haemophilus influenzae (NTHi).ConclusionOur findings demonstrated reduced expression of Siglec-1 on alveolar macrophages in COPD, which is involved in engulfment of NTHi.

Late Neutrophil Priming Following a Single Session of High-intensity Interval Exercise.

This study evaluated the effect of an acute high-intensity interval exercise (HIIE) session on the function of human neutrophils. Twelve sedentary men performed a HIIE session (8 bouts of 60 s at 90% of peak power, intercalated with 75 s of active recovery at 30 W). Neutrophils were collected before, 30 min and 24 h after the exercise session for the evaluation of phagocytic capacity, expression of phagocytic receptors, reactive oxygen species generation, and redox status. 24 h after the HIIE session, an increase was observed in both neutrophil phagocytic capacity and yeast-induced generation of reactive oxygen species, which indicates neutrophil priming in response to an acute HIIE session. Neutrophils also presented an increase in superoxide dismutase activity 24 h after the exercise. Improvement in neutrophil function was accompanied by increased serum levels of IL-8 and increased concentration of plasma lactate dehydrogenase. Our findings show a late activating effect of one HIIE session on neutrophils. We propose that priming of neutrophils by HIIE may play a role in skeletal muscle inflammation after exercise.

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

Small molecule–based inhibition of MEK1/2 proteins dampens inflammatory responses to malaria, reduces parasite load, and mitigates pathogenic outcomes

Malaria infections cause several systemic and severe single- or multi-organ pathologies, killing hundreds of thousands of people annually. Considering the existing widespread resistance of malaria parasites to anti-parasitic drugs and their high propensity to develop drug resistance, alternative strategies are required to manage malaria infections. Because malaria is a host immune response-driven disease, one approach is based on gaining a detailed understanding of the molecular and cellular processes that modulate malaria-induced innate and adaptive immune responses. Here, using a mouse cerebral malaria model and small-molecule inhibitors, we demonstrate that inhibiting MEK1/2, the upstream kinases of ERK1/2 signaling, alters multifactorial components of the innate and adaptive immune responses, controls parasitemia, and blocks pathogenesis. Specifically, MEK1/2 inhibitor treatment up-regulated B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic activity, enhancing parasite clearance by macrophages and neutrophils. Further, the MEK1/2 inhibitor treatment down-regulated pathogenic pro-inflammatory and helper T cell 1 (Th1) responses and up-regulated beneficial anti-inflammatory cytokine responses and Th2 responses. These inhibitor effects resulted in reduced granzyme B expression by T cells, chemokine and intracellular cell adhesion molecule 1 (ICAM-1) expression in the brain, and chemokine receptor expression by both myeloid and T cells. These bimodal effects of the MEK1/2 inhibitor treatment on immune responses contributed to decreased parasite biomass, organ inflammation, and immune cell recruitment, preventing tissue damage and death. In summary, we have identified several previously unrecognized immune regulatory processes through which a MEK1/2 inhibitor approach controls malaria parasitemia and mitigates pathogenic effects on host organs.

CD36 receptor regulates malaria-induced immune responses primarily at early blood stage infection contributing to parasitemia control and resistance to mortality

In malaria, CD36 plays several roles, including mediating parasite sequestration to host organs, phagocytic clearance of parasites, and regulation of immunity. Although the functions of CD36 in parasite sequestration and phagocytosis have been clearly defined, less is known about its role in malaria immunity. Here, to understand the function of CD36 in malaria immunity, we studied parasite growth, innate and adaptive immune responses, and host survival in WT and Cd36-/- mice infected with a non-lethal strain of Plasmodium yoelii Compared with Cd36-/- mice, WT mice had lower parasitemias and were resistant to death. At early but not at later stages of infection, WT mice had higher circulatory proinflammatory cytokines and lower anti-inflammatory cytokines than Cd36-/- mice. WT mice showed higher frequencies of proinflammatory cytokine-producing and lower frequencies of anti-inflammatory cytokine-producing dendritic cells (DCs) and natural killer cells than Cd36-/- mice. Cytokines produced by co-cultures of DCs from infected mice and ovalbumin-specific, MHC class II-restricted α/β (OT-II) T cells reflected CD36-dependent DC function. WT mice also showed increased Th1 and reduced Th2 responses compared with Cd36-/- mice, mainly at early stages of infection. Furthermore, in infected WT mice, macrophages and neutrophils expressed higher levels of phagocytic receptors and showed enhanced phagocytosis of parasite-infected erythrocytes than those in Cd36-/- mice in an IFN-γ-dependent manner. However, there were no differences in malaria-induced humoral responses between WT and Cd36-/- mice. Overall, the results show that CD36 plays a significant role in controlling parasite burden by contributing to proinflammatory cytokine responses by DCs and natural killer cells, Th1 development, phagocytic receptor expression, and phagocytic activity.

ERK Signaling-regulated Immune Responses that contributes to Malaria Parasitemia and Pathogenesis

Abstract Malaria morbidity and mortality represent a major health and social burdens in many countries around the globe. Given that malaria parasites are becoming more and more refractory to several commonly used drugs, a vaccine is needed to control malaria. However, development of an effective vaccine has been hampered due to incomplete understanding of immunological processes and their regulation. We studied the role of ERK1/2 MAPK signaling regulated immune responses by targeting the signaling pathway in P. berghei ANKA-infected mice, an experimental cerebral malaria model. The signaling was targeted using two small molecule inhibitors PD98059 and an FDA approved anti-cancer drug trametinib, both of which specifically inhibit activities of MEK1/MEK2, the upstream kinases of ERK1/2. The results showed that ERK signaling significantly curtails B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic clearance of parasites, thereby contributing to high parasite burden. The results also showed that ERK1/2 signaling upregulates pro-inflammatory responses and downregulates anti-inflammatory responses, promoting Th1 response and impeding Th2 response. This resulted in increased T cell cytotoxicity and enhanced infiltration of immune cells to the brain, brain vascular damage and dysfunction. Thus, we identified several previously unrecognized immune regulatory processes through which ERK1/2 signaling exacerbates malaria parasitemia and pathogenesis. Our findings highlight how the ERK1/2 signaling-regulated immune responses limit parasite clearance and contribute to severe malaria pathogenesis.

CD36 regulates malaria-induced innate immune responses

Abstract Malaria causes enormous morbidity and mortality worldwide. However, an effective vaccine is not yet available due to the knowledge gap in various molecular and cellular processes involved in protective immunity development. CD36, a multifunctional scavenger receptor, plays several important roles in malaria, including sequestration of parasites in host organs, parasitemia control, and protective immunity. Although the role of CD36 in the former two processes has been well understood, less is known about the function of CD36 in the latter. We studied the role of CD36 in host survival and immune responses in non-lethal Plasmodium yoelii infected WT and CD36−/− mice. WT mice showed lower parasitemia and higher survival rate compared to the CD36−/− mice. At early but not at later stages of infection, the circulatory pro-inflammatory cytokines were higher and anti-inflammatory cytokines were lower in WT mice than in CD36−/− mice. DCs, NK and T cells of WT mice had higher frequencies of pro-inflammatory and lower frequencies of anti-inflammatory cytokine-producing cells than CD36−/− mice. Additionally, Th1 response was decreased, whereas Th2 response was increased in CD36−/− mice. Compared to CD36−/− mice, macrophages and neutrophils of WT mice showed higher phagocytic activity and phagocytic receptors expression in an IFN-g dependent manner. However no difference in the malaria-induced humoral responses between WT and CD36−/− mice was observed. Our data demonstrated that CD36 plays an important role in parasitemia control and protective immunity by upregulating pro-inflammatory cytokine responses by DCs and NK cells, promoting Th1 response, and contributing to phagocytic receptor expression and phagocytic activity.

Macrophage Polarization Modulates FcγR- and CD13-Mediated Phagocytosis and Reactive Oxygen Species Production, Independently of Receptor Membrane Expression.

In response to microenvironmental cues, macrophages undergo a profound phenotypic transformation acquiring distinct activation phenotypes ranging from pro-inflammatory (M1) to anti-inflammatory (M2). To study how activation phenotype influences phagocytosis and production of reactive oxygen species (ROS) mediated by receptors for IgG antibodies (Fcγ receptors) and by CD13, human monocyte-derived macrophages were polarized to distinct phenotypes using IFN-γ (Mϕ-IFN-γ), IL-4 (Mϕ-IL-4), or IL-10 (Mϕ-IL-10). Phenotypically, Mϕ-IFN-γ were characterized as CD14+CD80+CD86+ cells, Mϕ-IL-4 as CD209highCD206+CD11b+CD14low, and Mϕ-IL-10 as CD16+CD163+ cells. Compared to non-polarized macrophages, FcγRI expression increased in Mϕ-IFN-γ and Mϕ-IL-10 and FcγRIII expression increased in Mϕ-IL-10. None of the polarizing cytokines modified FcγRII or CD13 expression. Functionally, we found that cytokine-mediated activation significantly and distinctively affected FcγR- and CD13-mediated phagocytosis and ROS generation. Compared to non-polarized macrophages, FcγRI-, FcγRII-, and CD13-mediated phagocytosis was significantly increased in Mϕ-IL-10 and decreased in Mϕ-IFN-γ, although both cytokines significantly upregulated FcγRI expression. IL-10 also increased phagocytosis of Escherichia coli, showing that the effect of IL-10 on macrophage phagocytosis is not specific for a particular receptor. Interestingly, Mϕ-IL-4, which showed poor FcγR- and CD13-mediated phagocytosis, showed very high phagocytosis of E. coli and zymosan. Coupled with phagocytosis, macrophages produce ROS that contribute to microbial killing. As expected, Mϕ-IFN-γ showed significant production of ROS after FcγRI-, FcγRII-, or CD13-mediated phagocytosis. Unexpectedly, we found that Mϕ-IL-10 can also produce ROS after simultaneous stimulation through several phagocytic receptors, as coaggregation of FcγRI/FcγRII/CD13 induced a belated but significant ROS production. Together, these results demonstrate that activation of macrophages by each cytokine distinctly modulates expression of phagocytic receptors, FcγR- and CD13-mediated phagocytosis, and ROS production.

Low-level arsenic causes chronic inflammation and suppresses expression of phagocytic receptors.

The impact of chronic low-level groundwater arsenic (As) exposure [in the range above the WHO-recommended limit of 10 μg/L but ≤50μg/L (permissible limit of As for many Asian countries)] was investigated for cross talk of inflammatory changes and expression of phagocytic receptors of exposed rural women (N, 45) from districts of 24 Parganas (south) and in matched control groups (N, 43) [As ≤10μg/L] from the same district. Systemic inflammation was evident from the upregulated levels of pro-inflammatory mediators like tumor necrosis factor-α (TNF-α); interleukins (ILs) like IL-6, IL-8, and IL-12; and C-reactive protein (CRP) in the sera and upregulated expression of protein kinase B phosphorylated at ser473 (pAKTser473)/nuclear factor-κB (NF-κB)/TNF-α axis in the leukocytes of exposed women with respect to control. We found that low-dose As exposure apart from inflicting inflammation altered the expression of phagocytic receptors-Fcγ receptors (FcγRs) and complement receptors (CRs). The leukocytes of the low-As-exposed women exhibited suppression of CD64, CD35, and CD11b and increased expression of CD16 with respect to control. Groundwater As showed a negative correlation with CD64 expression on monocytes [Pearson's r, -0.8205; 95% confidence interval (CI), -0.8789 to -0.7379] and granulocytes [r, -0.7635; 95% CI, -0.8388 to -0.6595] and a positive correlation with CD16 on granulocytes [r, 0.8363; 95% CI, 0.7599 to 0.8899]. A negative correlation of groundwater As was also observed with expression of CD35 on granulocytes [r, -0.8780; 95% CI, -0.9185 to -0.8192] and monocytes [r, -0.7778; 95% CI, -0.8490 to -0.6790] and CD11b on monocytes [r, -0.6035; 95% CI, -0.7218 to -0.4511]. Therefore, it may be indicated that chronic low-level As exposure (11-50μg/L) not only evoked chronic inflammatory changes but also suppressed the expression of FcγRs and CRs in the exposed women. This, in turn, may lead to susceptibility towards pathogenic infections or in long run may even contribute towards chronic inflammatory diseases including cancer.

Pseudomonas aeruginosa promotes autophagy to suppress macrophage-mediated bacterial eradication

ObjectivesTo explore the role of autophagy on macrophage-mediated phagocytosis and intracellular killing of Pseudomonas aeruginosa (PA), a common extracellular bacterium which often causes various opportunistic infections. MethodsMacrophages were infected with PA or stimulated with zymosan bioparticles. Autophagy was tested by fluorescent microscopy and Western blot for LC3. Phagocytosis and killing efficiency were assessed by plate count assay, flow cytometry or immunofluorescent staining. Phagocytic receptor expression, ROS generation and NO production were examined by PCR, flow cytometry and Griess reaction, respectively. ResultsPA infection induced autophagy activation in both mouse and human macrophages. Induction of autophagy by rapamycin or starvation significantly inhibited PA internalization by downregulating phagocytosis receptor expression, and suppressed intracellular killing of PA via reducing ROS and NO production in macrophages. While knockdown of autophagy molecules ATG7 or Beclin1 enhanced macrophage-mediated phagocytosis and intracellular killing of PA. Additionally, confocal microscopy data showed that induction of autophagy reduced the number of phagosomes and phagolysosomes in macrophages after stimulation with zymosan bioparticles. ConclusionsOur study suggested that PA promotes autophagy to suppress macrophage-mediated bacterial phagocytosis and intracellular killing. These insights demonstrated a novel immune evasion mechanism employed by PA, which may provide potential therapeutic strategies of PA infectious diseases.

Phenylbutyrate Is Bacteriostatic against Mycobacterium tuberculosis and Regulates the Macrophage Response to Infection, Synergistically with 25-Hydroxy-Vitamin D3.

Adjunctive vitamin D treatment for pulmonary tuberculosis enhances resolution of inflammation but has modest effects on bacterial clearance. Sodium 4-phenylbutyrate (PBA) is in clinical use for a range of conditions and has been shown to synergise with vitamin D metabolites to upregulate cathelicidin antimicrobial peptide (CAMP) expression. We investigated whether clinically attainable plasma concentrations of PBA (0.4-4mM) directly affect Mycobacterium tuberculosis (Mtb) growth and human macrophage and PBMC response to infection. We also tested the ability of PBA to enhance the immunomodulatory actions of the vitamin D metabolite 25(OH)D3 during infection and synergistically inhibit intracellular Mtb growth. PBA inhibited Mtb growth in broth with an MIC99 of 1mM, which was reduced to 0.25mM by lowering pH. During human macrophage infection, PBA treatment restricted Mtb uptake, phagocytic receptor expression and intracellular growth in a dose-dependent manner. PBA independently regulated CCL chemokine secretion and induced expression of the antimicrobial LTF (lactoferrin), the anti-inflammatory PROC (protein C) and multiple genes within the NLRP3 inflammasome pathway. PBA co-treatment with 25(OH)D3 synergistically modulated expression of numerous vitamin D-response genes, including CAMP, CYP24A1, CXCL10 and IL-37. This synergistic effect was dependent on MAPK signalling, while the effect of PBA on LTF, PROC and NLRP3 was MAPK-independent. During PBA and 25(OH)D3 co-treatment of human macrophages, in the absence of exogenous proteinase 3 (PR3) to activate cathelicidin, Mtb growth restriction was dominated by the effect of PBA, while the addition of PR3 enhanced growth restriction by 25(OH)D3 and PBA co-treatment. This suggests that PBA augments vitamin D–mediated cathelicidin-dependent Mtb growth restriction by human macrophages and independently induces antimicrobial and anti-inflammatory action. Therefore through both host-directed and bacterial-directed mechanisms PBA and vitamin D may prove an effective combinatorial adjunct therapy for tuberculosis to both resolve immunopathology and enhance bacterial clearance.

Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis

The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. Here we demonstrate that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, SIMU and DRPR. Moreover, we reveal that the phagocytic ability of embryonic glia is established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, we provide evidence for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of SIMU and DRPR specific expression. Taken together, our study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.

Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14++CD16+ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14++CD16+ monocyte subset.

Histone Deacetylase Inhibitors Impair Antibacterial Defenses of Macrophages

Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages.

Toll-Like Receptor 2- and MyD88-Dependent Phosphatidylinositol 3-Kinase and Rac1 Activation Facilitates the Phagocytosis ofListeria monocytogenesby Murine Macrophages

Toll-like receptors (TLRs) play a key role in the innate immune response by sensing bacterial ligands. The mechanisms involved in the TLR-mediated cytokine response are well established; however, the possible contribution of TLR-dependent recognition of bacteria to macrophage phagocytosis remains unclear. Listeria monocytogenes is an intracellular, parasitic, Gram-positive bacterium recognized mainly by TLR2. In this study, we investigated whether TLR2-dependent signaling is involved in the phagocytosis of L. monocytogenes by macrophages. We found no difference in the number of L. monocytogenes cells associating with wild-type (WT) and TLR2(-/-) macrophages 1 h after infection. However, the number of L. monocytogenes cells phagocytosed in TLR2(-/-) and MyD88(-/-) macrophages was significantly lower than that of WT macrophages. In addition, lipopolysaccharide (LPS) treatment restored impaired phagocytic activity of TLR2(-/-) macrophages but did not enhance the activity of MyD88(-/-) macrophages. The efficiency of phagocytosis was suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases but not by cycloheximide. Moreover, functional activation of PI3K and Rac1 was impaired in TLR2(-/-) and MyD88(-/-) macrophages. In an in vivo infection model, we found significantly lower numbers of L. monocytogenes cells phagocytosed in peritoneal macrophages of TLR2(-/-) and MyD88(-/-) mice after intraperitoneal infection. Moreover, a lower number of bacteria were detected in the spleens of TLR2(-/-) mice 1 day after intravenous infection than in WT mice. These results clearly indicated that TLR2-MyD88-dependent signaling enhances the basal level of phagocytosis of L. monocytogenes by macrophages through activation of PI3K and Rac1, not by synthesis of proinflammatory cytokines or expression of phagocytic receptors.

Age-related alterations in innate immune receptor expression and ability of macrophages to respond to pathogen challenge in vitro

The impact of ageing in innate immunity is poorly understood. Studies in the mouse model have described altered innate immune functions in aged macrophages, although these were not generally linked to altered expression of receptors or regulatory molecules. Moreover, the influence of ageing in the expression of these molecules has not been systematically examined. We investigated age-dependent expression differences in selected Toll-like and other pattern-recognition receptors, receptors involved in inflammatory amplification, and in transmembrane and intracellular regulators of inflammatory signaling. Young and aged macrophages were examined under resting conditions or upon activation with Porphyromonas gingivalis, a major pathogen in periodontal disease, the prevalence and severity of which increase in old age. We detected a limited number of age-dependent alterations, involving both reduction and increase of immune activity. Interestingly, surface expression of receptors that amplify inflammation (C5a anaphylatoxin receptor and triggering receptor expressed on myeloid cells [TREM]-1) was elevated in aged macrophages. No significant age-dependent differences were observed regarding the phagocytosis and intracellular killing of P. gingivalis, consistent with lack of significant changes in phagocytic receptor expression and induction of antimicrobial molecules. Therefore, at least at the cellular level, certain aspects of innate immune function may not necessarily decline with age.