Leukotrienes in Innate Immunity: Still Underappreciated after All These Years?

Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase pathway of arachidonate metabolism. Though best known for their role in asthma, they have broad actions that touch on virtually every aspect of mammalian biology. In a Brief Review published in the journal in 2005, we presented the existing evidence supporting a role for LTs in host defense. In this updated Brief Review, we focus on selected advances since then. We detail new insights into mechanisms and regulation of LT biosynthesis; the protective roles of LTs in the host response to diverse classes of pathogens, with an emphasis on viruses, including SARS-CoV-2; the phagocyte signal transduction mechanisms by which LTs exert their antimicrobial actions; the capacity for overexuberant LT production to promote tissue damage; and roles of LTs in the noninfectious immune-relevant conditions neuroinflammation and cancer.

Alveolar macrophage-derived extracellular vesicles inhibit endosomal fusion of influenza virus.

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.

Bortezomib Inhibits Lung Fibrosis and Fibroblast Activation without Proteasome Inhibition.

The U.S. Food and Drug Administration-approved proteasomal inhibitor bortezomib (BTZ) has attracted interest for its potential antifibrotic actions. However, neither its in vivo efficacy in lung fibrosis nor its dependence on proteasome inhibition has been conclusively defined. In this study, we assessed the therapeutic efficacy of BTZ in a mouse model of pulmonary fibrosis, developed an in vitro protocol to define its actions on diverse fibroblast activation parameters, determined its reliance on proteasome inhibition for these actions in vivo and in vitro, and explored alternative mechanisms of action. The therapeutic administration of BTZ diminished the severity of pulmonary fibrosis without reducing proteasome activity in the lung. In experiments designed to mimic this lack of proteasome inhibition in vitro, BTZ reduced fibroblast proliferation, differentiation into myofibroblasts, and collagen synthesis. It promoted dedifferentiation of myofibroblasts and overcame their characteristic resistance to apoptosis. Mechanistically, BTZ inhibited kinases important for fibroblast activation while inducing the expression of DUSP1 (dual-specificity protein phosphatase 1), and knockdown of DUSP1 abolished its antifibrotic actions in fibroblasts. Collectively, these findings suggest that BTZ exhibits a multidimensional profile of robust inhibitory actions on lung fibroblasts as well as antifibrotic actions in vivo. Unexpectedly, these actions appear to be independent of proteasome inhibition, instead attributable to the induction of DUSP1.

Antifibrotic factor KLF4 is repressed by the miR-10/TFAP2A/TBX5 axis in dermal fibroblasts: insights from twins discordant for systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is a complex disease of unknown aetiology in which inflammation and fibrosis lead to multiple organ damage. There is currently no effective therapy that can halt the progression of fibrosis or reverse it, thus studies that provide novel insights into disease pathogenesis and identify novel potential therapeutic targets are critically needed. METHODS: We used global gene expression and genome-wide DNA methylation analyses of dermal fibroblasts (dFBs) from a unique cohort of twins discordant for SSc to identify molecular features of this pathology. We validated the findings using in vitro, ex vivo and in vivo models. RESULTS: Our results revealed distinct differentially expressed and methylated genes, including several transcription factors involved in stem cell differentiation and developmental programmes (KLF4, TBX5, TFAP2A and homeobox genes) and the microRNAs miR-10a and miR-10b which target several of these deregulated genes. We show that KLF4 expression is reduced in SSc dFBs and its expression is repressed by TBX5 and TFAP2A. We also show that KLF4 is antifibrotic, and its conditional knockout in fibroblasts promotes a fibrotic phenotype. CONCLUSIONS: Our data support a role for epigenetic dysregulation in mediating SSc susceptibility in dFBs, illustrating the intricate interplay between CpG methylation, miRNAs and transcription factors in SSc pathogenesis, and highlighting the potential for future use of epigenetic modifiers as therapies.

Resident alveolar macrophage-derived vesicular SOCS3 dampens allergic airway inflammation.

Resident alveolar macrophages (AMs) suppress allergic inflammation in murine asthma models. Previously we reported that resident AMs can blunt inflammatory signaling in alveolar epithelial cells (ECs) by transcellular delivery of suppressor of cytokine signaling 3 (SOCS3) within extracellular vesicles (EVs). Here we examined the role of vesicular SOCS3 secretion as a mechanism by which AMs restrain allergic inflammatory responses in airway ECs. Bronchoalveolar lavage fluid (BALF) levels of SOCS3 were reduced in asthmatics and in allergen-challenged mice. Ex vivo SOCS3 secretion was reduced in AMs from challenged mice and this defect was mimicked by exposing normal AMs to cytokines associated with allergic inflammation. Both AM-derived EVs and synthetic SOCS3 liposomes inhibited the activation of STAT3 and STAT6 as well as cytokine gene expression in ECs challenged with IL-4/IL-13 and house dust mite (HDM) extract. This suppressive effect of EVs was lost when they were obtained from AMs exposed to allergic inflammation-associated cytokines. Finally, inflammatory cell recruitment and cytokine generation in the lungs of OVA-challenged mice were attenuated by intrapulmonary pretreatment with SOCS3 liposomes. Overall, AM secretion of SOCS3 within EVs serves as a brake on airway EC responses during allergic inflammation, but is impaired in asthma. Synthetic liposomes encapsulating SOCS3 can rescue this defect and may serve as a framework for novel therapeutic approaches targeting airway inflammation.

Distinctive Effects of GM-CSF and M-CSF on Proliferation and Polarization of Two Major Pulmonary Macrophage Populations.

GM-CSF is required for alveolar macrophage (AM) development shortly after birth and for maintenance of AM functions throughout life, whereas M-CSF is broadly important for macrophage differentiation and self-renewal. However, the comparative actions of GM-CSF and M-CSF on AMs are incompletely understood. Interstitial macrophages (IMs) constitute a second major pulmonary macrophage population. However, unlike AMs, IM responses to CSFs are largely unknown. Proliferation, phenotypic identity, and M1/M2 polarization are important attributes of all macrophage populations, and in this study, we compared their modulation by GM-CSF and M-CSF in murine primary AMs and IMs. CSFs increased the proliferation capacity and upregulated antiapoptotic gene expression in AMs but not IMs. GM-CSF, but not M-CSF, reinforced the cellular identity, as identified by surface markers, of both cell types. GM-CSF, but not M-CSF, increased the expression of both M1 and M2 markers exclusively in AMs. Finally, CSFs enhanced the IFN-γ- and IL-4-induced polarization ability of AMs but not IMs. These first (to our knowledge) data comparing effects on the two pulmonary macrophage populations demonstrate that the activating actions of GM-CSF and M-CSF on primary AMs are not conserved in primary IMs.

Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation.

BACKGROUND: Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4, are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. METHODS: Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2-mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes. RESULTS: Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated leukotriene B4 biosynthesis. Bmpr2-mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-ß) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-ß antagonism suggests that TGF-ß is critical for neointimal transformation. CONCLUSIONS: In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-ß signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.

Molecular determinants of mesenchymal cell activation in fibroproliferative diseases.

Uncontrolled scarring, or fibrosis, can interfere with the normal function of virtually all tissues of the body, ultimately leading to organ failure and death. Fibrotic diseases represent a major cause of death in industrialized countries. Unfortunately, no curative treatments for these conditions are yet available, highlighting the critical need for a better fundamental understanding of molecular mechanisms that may be therapeutically tractable. The ultimate indispensable effector cells responsible for deposition of extracellular matrix proteins that comprise scars are mesenchymal cells, namely fibroblasts and myofibroblasts. In this review, we focus on the biology of these cells and the molecular mechanisms that regulate their pertinent functions. We discuss key pro-fibrotic mediators, signaling pathways, and transcription factors that dictate their activation and persistence. Because of their possible clinical and therapeutic relevance, we also consider potential brakes on mesenchymal cell activation and cellular processes that may facilitate myofibroblast clearance from fibrotic tissue-topics that have in general been understudied.

Interleukin-36γ and IL-36 receptor signaling mediate impaired host immunity and lung injury in cytotoxic Pseudomonas aeruginosa pulmonary infection: Role of prostaglandin E2.

Pseudomonas aeruginosa is a Gram-negative pathogen that can lead to severe infection associated with lung injury and high mortality. The interleukin (IL)-36 cytokines (IL-36α, IL-36ß and IL-36γ) are newly described IL-1 like family cytokines that promote inflammatory response via binding to the IL-36 receptor (IL-36R). Here we investigated the functional role of IL-36 cytokines in the modulating of innate immune response against P. aeruginosa pulmonary infection. The intratracheal administration of flagellated cytotoxic P. aeruginosa (ATCC 19660) upregulated IL-36α and IL-36γ, but not IL-36ß, in the lungs. IL-36α and IL-36γ were expressed in pulmonary macrophages (PMs) and alveolar epithelial cells in response to P. aeruginosa in vitro. Mortality after bacterial challenge in IL-36 receptor deficient (IL-36R-/-) mice and IL-36γ deficient (IL-36γ-/-) mice, but not IL-36α deficient mice, was significantly lower than that of wild type mice. Decreased mortality in IL-36R-/- mice and IL-36γ-/- mice was associated with reduction in bacterial burden in the alveolar space, bacterial dissemination, production of inflammatory cytokines and lung injury, without changes in lung leukocyte influx. Interestingly, IL-36γ enhanced the production of prostaglandin E2 (PGE2) during P. aeruginosa infection in vivo and in vitro. Treatment of PMs with recombinant IL-36γ resulted in impaired bacterial killing via PGE2 and its receptor; EP2. P. aeruginosa infected EP2 deficient mice or WT mice treated with a COX-2-specific inhibitor showed decreased bacterial burden and dissemination, but no change in lung injury. Finally, we observed an increase in IL-36γ, but not IL-36α, in the airspace and plasma of patients with P. aeruginosa-induced acute respiratory distress syndrome. Thus, IL-36γ and its receptor signal not only impaired bacterial clearance in a possible PGE2 dependent fashion but also mediated lung injury during P. aeruginosa infection.

The Induction of Pro-IL-1ß by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production.

PGE2 has been shown to increase the transcription of pro-IL-1ß. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1ß by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE2 on IL-1ß production. We have found that in murine bone marrow-derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1ß transcription, as well as boosting the ability of LPS to induce IL-1ß mRNA and pro-IL-1ß while inhibiting the production of TNF-α. This results in an increase in mature IL-1ß production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1ß production. By blocking PGE2 production with indomethacin, we made a striking finding that endogenous PGE2 is essential for LPS-induced pro-IL-1ß production, suggesting a positive feedback loop. The effect of endogenous PGE2 was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1ß, PGE2 boosted LPS-induced IL-1ß production. PGE2 did not inhibit ATP-induced mature IL-1ß production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1ß, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1ß and back to PGE2, which itself is induced by IL-1ß, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE2 on TNF-α production.

Mechanisms and modulation of microvesicle uptake in a model of alveolar cell communication.

Extracellular vesicles, including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which extracellular vesicles are internalized is incompletely characterized, it is generally considered to involve endocytosis and an initial surface-binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here, we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage-derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptor-dependent endocytosis and is subject to bidirectional modulation by relevant pathologic perturbations.

Ablation of the leptin receptor in myeloid cells impairs pulmonary clearance of Streptococcus pneumoniae and alveolar macrophage bactericidal function.

Leptin is a pleiotropic hormone produced by white adipose tissue that regulates appetite and many physiological functions, including the immune response to infection. Genetic leptin deficiency in humans and mice impairs host defenses against respiratory tract infections. Since leptin deficiency is associated with obesity and other metabolic abnormalities, we generated mice that lack the leptin receptor (LepRb) in cells of the myeloid linage (LysM-LepRb-KO) to evaluate its impact in lean metabolically normal mice in a murine model of pneumococcal pneumonia. We observed higher lung and spleen bacterial burdens in LysM-LepRb-KO mice following an intratracheal challenge with Streptococcus pneumoniae. Although numbers of leukocytes recovered from bronchoalveolar lavage fluid did not differ between groups, we did observe higher levels of pulmonary IL-13 and TNFα in LysM-LepRb-KO mice 48 h post infection. Phagocytosis and killing of ingested S. pneumoniae were also impaired in alveolar macrophages (AMs) from LysM-LepRb-KO mice in vitro and were associated with reduced LTB4 and enhanced PGE2 synthesis in vitro. Pretreatment of AMs with LTB4 and the cyclooxygenase inhibitor, indomethacin, restored phagocytosis but not bacterial killing in vitro. These results confirm our previous observations in leptin-deficient ( ob/ob) and fasted mice and demonstrate that decreased leptin action, as opposed to metabolic irregularities associated with obesity or starvation, is responsible for the defective host defense against pneumococcal pneumonia. They also provide novel targets for therapeutic intervention in humans with bacterial pneumonia.

Cysteinyl leukotrienes as novel host factors facilitating Cryptococcus neoformans penetration into the brain.

Cryptococcus neoformas infection of the central nervous system (CNS) continues to be an important cause of mortality and morbidity, and a major contributing factor is our incomplete knowledge of the pathogenesis of this disease. Here, we provide the first direct evidence that C. neoformans exploits host cysteinyl leukotrienes (LTs), formed via LT biosynthetic pathways involving cytosolic phospholipase A2 α (cPLA2 α) and 5-lipoxygenase (5-LO) and acting via cysteinyl leukotriene type 1 receptor (CysLT1), for penetration of the blood-brain barrier. Gene deletion of cPLA2 α and 5-LO and pharmacological inhibition of cPLA2 α, 5-LO and CysLT1 were effective in preventing C. neoformans penetration of the blood-brain barrier in vitro and in vivo. A CysLT1 antagonist enhanced the efficacy of an anti-fungal agent in therapy of C. neoformans CNS infection in mice. These findings demonstrate that host cysteinyl LTs, dependent on the actions of cPLA2 α and 5-LO, promote C. neoformans penetration of the blood-brain barrier and represent novel targets for elucidating the pathogenesis and therapeutic development of C. neoformans CNS infection.

Alveolar Epithelial Cell-Derived Prostaglandin E2 Serves as a Request Signal for Macrophage Secretion of Suppressor of Cytokine Signaling 3 during Innate Inflammation.

Preservation of gas exchange mandates that the pulmonary alveolar surface restrain unnecessarily harmful inflammatory responses to the many challenges to which it is exposed. These responses reflect the cross-talk between alveolar epithelial cells (AECs) and resident alveolar macrophages (AMs). We recently determined that AMs can secrete suppressor of cytokine signaling (SOCS) proteins within microparticles. Uptake of these SOCS-containing vesicles by epithelial cells inhibits cytokine-induced STAT activation. However, the ability of epithelial cells to direct AM release of SOCS-containing vesicles in response to inflammatory insults has not been studied. In this study, we report that SOCS3 protein was elevated in bronchoalveolar lavage fluid of both virus- and bacteria-infected mice, as well as in an in vivo LPS model of acute inflammation. In vitro studies revealed that AEC-conditioned medium (AEC-CM) enhanced AM SOCS3 secretion above basal levels. Increased amounts of PGE2 were present in AEC-CM after LPS challenge, and both pharmacologic inhibition of PGE2 synthesis in AECs and neutralization of PGE2 in AEC-CM implicated this prostanoid as the major AEC-derived factor mediating enhanced AM SOCS3 secretion. Moreover, pharmacologic blockade of PGE2 synthesis or genetic deletion of a PGE2 synthase similarly attenuated the increase in bronchoalveolar lavage fluid SOCS3 noted in lungs of mice challenged with LPS in vivo. These results demonstrate a novel tunable form of cross-talk in which AECs use PGE2 as a signal to request SOCS3 from AMs to dampen their endogenous inflammatory responses during infection.

Distinct PKA regulatory subunits mediate PGE2 inhibition of TGFß-1-stimulated collagen I translation and myofibroblast differentiation.

Prostaglandin E2 (PGE2), via cAMP signaling, inhibits a variety of fibroblast functions relevant to fibrogenesis. Among these are their translation of collagen I protein and their differentiation to myofibroblasts. PKA is central to these actions, with cAMP binding to regulatory (R) subunits leading to the release of catalytic subunits. Here we examined the role of specific PKAR subunit isoforms in these inhibitory actions in transforming growth factor ß-1 (TGFß-1)-stimulated human lung fibroblasts (HLFs). HLFs expressed all four R subunit isoforms. siRNA-mediated knockdown of subunits PKARIα and PKARIIα had no effect on PGE2 inhibition of either process. However, knockdown of PKARIß selectively attenuated PGE2 inhibition of collagen I protein expression, whereas knockdown of PKARIIß selectively attenuated PGE2 inhibition of expression of the myofibroblast differentiation marker, α-smooth muscle actin (α-SMA). cAMP analogs that selectively activate either PKARIß or PKARIIß exclusively inhibited collagen I synthesis or differentiation, respectively. In parallel, the PKARIß agonist (but not a PKARIIß agonist) reduced phosphorylation of two proteins involved in protein translation, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). By contrast, the PKARIIß agonist (but not a PKARIß agonist) reduced levels of the differentiation-associated phosphorylated focal adhesion kinase (p-FAK) as well as the relative mRNA and protein expression of serum response factor (SRF), a transcription factor necessary for myofibroblast differentiation. Our results demonstrate that cAMP inhibition of collagen I translation and myofibroblast differentiation reflects the actions of distinct PKAR subunits.

Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-ß and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome, hydroxyproline, collagen type I α 1 chain, and collagen type I α 2 chain). Pharmacologic or RNA interference-specific interventions suppressed complement activation (C3a and C5a) and soluble terminal complement complex formation (C5b-9) locally and active TGF-ß1 systemically. C3aR/C5aR antagonists suppressed local mRNA expressions of tgfb2, tgfbr1/2, ltbp1/2, serpine1, tsp1, bmp1/4, pdgfbb, igf1, but restored the proteoglycan, dcn Clinically, compared with pathologically normal human subjects, patients with IPF presented local induction of C5aR, local and systemic induction of soluble C5b-9, and amplified expression of C3aR/C5aR in lesions. The blockade of C3aR and C5aR arrested the progression of fibrosis by attenuating local complement activation and TGF-ß/bone morphologic protein signaling as well as restoring decorin, which suggests a promising therapeutic strategy for patients with IPF.-Gu, H., Fisher, A. J., Mickler, E. A., Duerson, F., III, Cummings, O. W., Peters-Golden, M., Twigg, H. L., III, Woodruff, T. M., Wilkes, D. S., Vittal, R. Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis.

Alveolar Macrophages in Allergic Asthma: the Forgotten Cell Awakes.

PURPOSE OF REVIEW: The role of alveolar macrophages in innate immune responses has long been appreciated. Here, we review recent studies evaluating the participation of these cells in allergic inflammation. RECENT FINDINGS: Immediately after allergen exposure, monocytes are rapidly recruited from the bloodstream and serve to promote acute inflammation. By contrast, resident alveolar macrophages play a predominantly suppressive role in an effort to restore homeostasis. As inflammation becomes established after repeated exposures, alveolar macrophages can polarize across a continuum of activation phenotypes, losing their suppressive functions and gaining pathogenic functions. Future research should focus on the diverse roles of monocytes/macrophages during various types and phases of allergic inflammation. These properties could lead us to new therapeutic opportunities.

Short-Term Regulation of FcγR-Mediated Phagocytosis by TLRs in Macrophages: Participation of 5-Lipoxygenase Products.

TLRs recognize a broad spectrum of microorganism molecules, triggering a variety of cellular responses. Among them, phagocytosis is a critical process for host defense. Leukotrienes (LTs), lipid mediators produced from 5-lipoxygenase (5-LO) enzyme, increase FcγR-mediated phagocytosis. Here, we evaluated the participation of TLR2, TLR3, TLR4, and TLR9 in FcγR-mediated phagocytosis and whether this process is modulated by LTs. Rat alveolar macrophages (AMs), murine bone marrow-derived macrophages (BMDMs), and peritoneal macrophages (PMs) treated with TLR2, TLR3, and TLR4 agonists, but not TLR9, enhanced IgG-opsonized sheep red blood cell (IgG-sRBC) phagocytosis. Pretreatment of AMs or BMDMs with drugs that block LT synthesis impaired the phagocytosis promoted by TLR ligands, and TLR potentiation was also abrogated in PMs and BMDMs from 5-LO-/- mice. LTB4 production induced by IgG engagement was amplified by TLR ligands, while cys-LTs were amplified by activation of TLR2 and TLR4, but not by TLR3. We also noted higher ERK1/2 phosphorylation in IgG-RBC-challenged cells when preincubated with TLR agonists. Furthermore, ERK1/2 inhibition by PD98059 reduced the phagocytic activity evoked by TLR agonists. Together, these data indicate that TLR2, TLR3, and TLR4 ligands, but not TLR9, amplify IgG-mediated phagocytosis by a mechanism which requires LT production and ERK-1/2 pathway activation.

Reversal of the Transcriptome by Prostaglandin E2 during Myofibroblast Dedifferentiation.

Myofibroblasts, the major effector cells in pathologic fibrosis, derive from the differentiation of fibroblasts driven by mediators such as transforming growth factor-ß1 (TGF-ß1) and biomechanical signals. Although the myofibroblast has traditionally been considered a terminally differentiated cell, the lipid mediator prostaglandin E2 (PGE2) has been shown to not only prevent but also reverse myofibroblast differentiation, as characterized by the ability of PGE2 to diminish expression of collagen I and α-smooth muscle actin in established myofibroblasts. Here, we use microarrays to examine the extent of transcriptomic changes that occur during TGF-ß1-induced differentiation and PGE2-induced dedifferentiation of myofibroblasts. Normal primary human adult lung fibroblasts were cultured for 24 hours with or without TGF-ß1 and treated for 48 hours with PGE2. Gene expression levels were assessed from total RNA on the Affymetrix U219 microarray. TGF-ß1 up-regulated 588 genes and down-regulated 689 genes compared with control cells. PGE2 reversed the expression of 363 (62%) of the TGF-ß1-up-regulated genes and 345 (50%) of the TGF-ß1-down-regulated genes. Genes up-regulated by TGF-ß1 and reversed by PGE2 were enriched in annotations for Cell Adhesion, Contractile Fiber, and Actin Binding, whereas genes down-regulated by TGF-ß1 but subsequently reversed by PGE2 were enriched in annotations for Glycoprotein, Polysaccharide Binding, and Regulation of Cell Migration. Surprisingly, the genes whose expression was affected by PGE2 differed between TGF-ß1-induced myofibroblasts and undifferentiated fibroblasts. These data demonstrate the capacity of PGE2 to effect marked global alterations in the transcriptomic program of differentiated myofibroblasts and emphasize the considerable plasticity of these cells.