Human cathelicidin (LL-37) is a defense peptide with antimicrobial activity against various pathogens. However, LL-37 can also trigger tissue injury by binding to host cell membranes. The cytotoxic ...effects of LL-37 may be especially relevant in chronic respiratory diseases characterized by increased LL-37. The aim of this study was to investigate whether the human collectin SP-A and a trimeric recombinant fragment thereof (rfhSP-A) can regulate the activities of LL-37. To this end, we studied the interaction of LL-37 with SP-A and rfhSP-A by intrinsic fluorescence, dynamic light scattering, and circular dichroism, as well as the effects of these proteins on the antimicrobial and cytotoxic activities of LL-37. Both SP-A and rfhSP-A bound LL-37 with high affinity at physiological ionic strength (
K
D
= 0.45 ± 0.01 nM for SP-A and 1.22 ± 0.7 nM for rfhSP-A). Such interactions result in the reduction of LL-37-induced cell permeability and IL-8 release in human pneumocytes, mediated by P2X7 channels. Binding of LL-37 to SP-A did not modify the properties of SP-A or the antibacterial activity of LL-37 against respiratory pathogens (
Klebsiella pneumoniae
,
Pseudomonas aeruginosa
, and nontypeable
Haemophilus influenzae
). SP-A/LL-37 complexes showed a greater ability to aggregate LPS vesicles than LL-37, which reduces endotoxin bioactivity. These results reveal the protective role of native SP-A in controlling LL-37 activities and suggest a potential therapeutic effect of rfhSP-A in reducing the cytotoxic and inflammatory actions of LL-37, without affecting its microbicidal activity against Gram-negative pathogens.
Activation of tissue repair program in macrophages requires the integration of IL-4/IL-13 cytokines and tissue-specific signals. In the lung, surfactant protein A (SP-A) is a tissue factor that ...amplifies IL-4Rα-dependent alternative activation and proliferation of alveolar macrophages (AMs) through the myosin18A receptor. However, the mechanism by which SP-A and IL-4 synergistically increase activation and proliferation of AMs is unknown. Here we show that SP-A amplifies IL-4-mediated phosphorylation of STAT6 and Akt by binding to myosin18A. Blocking PI3K activity or the myosin18A receptor abrogates SP-A´s amplifying effects on IL-4 signaling. SP-A alone activates Akt, mTORC1, and PKCζ and inactivates GSK3α/β by phosphorylation, but it cannot activate arginase-1 activity or AM proliferation on its own. The combined effects of IL-4 and SP-A on the mTORC1 and GSK3 branches of PI3K-Akt signaling contribute to increased AM proliferation and alternative activation, as revealed by pharmacological inhibition of Akt (inhibitor VIII) and mTORC1 (rapamycin and torin). On the other hand, the IL-4+SP-A-driven PKCζ signaling axis appears to intersect PI3K activation with STAT6 phosphorylation to achieve more efficient alternative activation of AMs. Consistent with IL-4+SP-A-driven activation of mTORC1 and mTORC2, both agonists synergistically increased mitochondrial respiration and glycolysis in AMs, which are necessary for production of energy and metabolic intermediates for proliferation and alternative activation. We conclude that SP-A signaling in AMs activates PI3K-dependent branched pathways that amplify IL-4 actions on cell proliferation and the acquisition of AM effector functions.
Abstract
Alveolar macrophages (AMs) are less able to respond to IL-4 in vivo than macrophages from the peritoneal cavity, due to a still-unknown factor of the lung environment. The aim of this study ...is to investigate whether surfactant lipids, which are continuously endocytosed by AMs, could influence IL-4-mediated alternative activation and proliferation of AMs. To that end, AMs were preincubated with surfactant lipids and stimulated with IL-4 in the presence or absence of surfactant protein SP-A, an amplifier of IL-4 actions. We found that alveolar lipids reduced IL-4- and IL-4+SP-A-dependent arginase activity, the expression of genes associated with alternative activation, and proliferation of AMs. Mechanistically, endocytosed lipids decreased IL-4- and IL-4+SP-A-induced activation of the PI3K-Akt-mTORC1 signaling axis, but not the IL-4-dependent STAT6 axis. Lipid-dependent inhibition of the Akt/mTORC1 signaling axis is consistent with reduced IL-4+SP-A-driven glycolysis and mitochondrial respiration as well as decreased ATP citrate lyase expression and histone acetylation stimulated by IL-4. We conclude that surfactant lipids inhibit PI3K-dependent signaling pathways and suggest that decrease of surfactant lipids in chronic lung diseases might augment IL-4-dependent fibrotic responses.
This research is funded by the Spanish Ministry of Science and Innovation through Grant PID2021-123044OB-I00 to C. Casals
Non-typeable
(NTHi) causes persistent respiratory infections in patients with chronic obstructive pulmonary disease (COPD), probably linked to its capacity to invade and reside within pneumocytes. In ...the alveolar fluid, NTHi is in contact with pulmonary surfactant, a lipoprotein complex that protects the lung against alveolar collapse and constitutes the front line of defense against inhaled pathogens and toxins. Decreased levels of surfactant phospholipids have been reported in smokers and patients with COPD. The objective of this study was to investigate the effect of surfactant phospholipids on the host-pathogen interaction between NTHi and pneumocytes. For this purpose, we used two types of surfactant lipid vesicles present in the alveolar fluid: (i) multilamellar vesicles (MLVs, > 1 μm diameter), which constitute the tensioactive material of surfactant, and (ii) small unilamellar vesicles (SUVs, 0.1 μm diameter), which are generated after inspiration/expiration cycles, and are endocytosed by pneumocytes for their degradation and/or recycling. Results indicated that extracellular pulmonary surfactant binds to NTHi, preventing NTHi self-aggregation and inhibiting adhesion of NTHi to pneumocytes and, consequently, inhibiting NTHi invasion. In contrast, endocytosed surfactant lipids, mainly via the scavenger receptor SR-BI, did not affect NTHi adhesion but inhibited NTHi invasion by blocking bacterial uptake in pneumocytes. This blockade was made possible by inhibiting Akt phosphorylation and Rac1 GTPase activation, which are signaling pathways involved in NTHi internalization. Administration of the hydrophobic fraction of lung surfactant
accelerated bacterial clearance in a mouse model of NTHi pulmonary infection, supporting the notion that the lipid component of lung surfactant protects against NTHi infection. These results suggest that alterations in surfactant lipid levels in COPD patients may increase susceptibility to infection by this pathogen.
Progressive myoclonus epilepsy of Lafora type (LD, MIM 254780) is a fatal autosomal recessive disorder characterized by the presence of progressive neurological deterioration, myoclonus, epilepsy and ...polyglucosan intracellular inclusion bodies, called Lafora bodies. Lafora bodies resemble glycogen with reduced branching, suggesting an alteration in glycogen metabolism. Linkage analysis and homozygosity mapping localized EPM2A, a major gene for LD, to chromosome 6q24. EPM2A encodes a protein of 331 amino acids (named laforin) with two domains, a dual-specificity phosphatase domain and a carbohydrate binding domain. Here we show that, in addition, laforin interacts with itself and with the glycogen targeting regulatory subunit R5 of protein phosphatase 1 (PP1). R5 is the human homolog of the murine Protein Targeting to Glycogen, a protein that also acts as a molecular scaffold assembling PP1 with its substrate, glycogen synthase, at the intracellular glycogen particles. The laforin–R5 interaction was confirmed by pull-down and co-localization experiments. Full-length laforin is required for the interaction. However, a minimal central region of R5 (amino acids 116–238), including the binding sites for glycogen and for glycogen synthase, is sufficient to interact with laforin. Point-mutagenesis of the glycogen synthase-binding site completely blocked the interaction with laforin. The majority of the EPM2A missense mutations found in LD patients result in lack of phosphatase activity, absence of binding to glycogen and lack of interaction with R5. Interestingly, we have found that the LD-associated EPM2A missense mutation G240S has no effect on the phosphatase or glycogen binding activities of laforin but disrupts the interaction with R5, suggesting that binding to R5 is critical for the laforin function. These results place laforin in the context of a multiprotein complex associated with intracellular glycogen particles, reinforcing the concept that laforin is involved in the regulation of glycogen metabolism.
Lafora progressive myoclonus epilepsy (LD) is a fatal autosomal recessive neurodegenerative disorder characterized by the presence of glycogen-like intracellular inclusions called Lafora bodies. LD ...is caused by mutations in two genes, EPM2A and EPM2B, encoding respectively laforin, a dual-specificity protein phosphatase, and malin, an E3 ubiquitin ligase. Previously, we and others have suggested that the interactions between laforin and PTG (a regulatory subunit of type 1 protein phosphatase) and between laforin and malin are critical in the pathogenesis of LD. Here, we show that the laforin–malin complex downregulates PTG-induced glycogen synthesis in FTO2B hepatoma cells through a mechanism involving ubiquitination and degradation of PTG. Furthermore, we demonstrate that the interaction between laforin and malin is a regulated process that is modulated by the AMP-activated protein kinase (AMPK). These findings provide further insights into the critical role of the laforin–malin complex in the control of glycogen metabolism and unravel a novel link between the energy sensor AMPK and glycogen metabolism. These data advance our understanding of the functional role of laforin and malin, which hopefully will facilitate the development of appropriate LD therapies.
Abstract
There is evidence that lung surfactant protein A (SP-A) plays an important role in modulating inflammation during lung infection. However, it has been reported that SP-A either stimulates or ...inhibits the inflammatory activity of IFN-γ on alveolar macrophages (aMϕ). The aim of this study was to determine the effect of human SP-A on alveolar macrophages stimulated with IFN-γ in the presence or absence of LPS. To accomplish this, purified rat aMϕ were cultured in the presence or absence of IFN-γ (10 ng/ml), LPS (1 ng/ml), SP-A (5, 12.5, 25 μg/mL), and combinations thereof. We measured iNOS synthesis, TNF-α secretion, as well as STAT-1, ERK, and Akt phosphorylation by rat aMϕ. We found that SP-A inhibited STAT1 phosphorylation activated by IFN-γ. In addition, SP-A diminished LPS-induced ERK and Akt phosphorylation. Finally, SP-A inhibited LPS+IFN-γ-induced TNF-α and iNOS production. Therefore, the inhibitory effect of SP-A on IFN-γ+LPS-elicited macrophage activation is due to SP-A attenuation of both inflamatory agents. To determine the mechanism by which SP-A abrogates IFN-γ effects, we studied the potential interaction between SP-A and IFN-γ. SP-A specifically bound to IFN-γ, with Kd = 28, 2 ± 8 nM. These data support the biological function of SP-A in host defense, revealing a role of this alveolar protein in limiting classical activation of aMϕ elicited by IFN-γ + LPS.
Abstract
It is well established that IL-4 induces alternative activation of macrophages involving intracellular activation of STAT6. Furthermore, it has been suggested that this cytokine has a ...proliferative effect on macrophages. On the other hand, there is evidence that pulmonary surfactant protein A (SP-A) plays an important role in modulating inflammatory response of alveolar macrophages (aMϕ). However, little is known about the effect of SP-A on the alternative activation of aMϕ. The aim of this study was to determine the effect of SP-A on aMϕ stimulated with IL-4. To accomplish this, purified rat aMϕ were stimulated with IL-4, different doses of SP-A, and combinations thereof. We measured arginase activity, cell viability, and STAT6 phosphorylation. We found that SP-A increased both IL-4-induced arginase activity and STAT6 phosphorylation. Finally, we observed more viable aMϕ in the presence of IL-4 and SP-A. We conclude that SP-A has a synergistic effect with IL-4 to lead aMϕ to an alternative activation. Furthermore, SP-A together with IL-4 may have either the protective effect of preventing aMϕ death or may cause cellular proliferation. Ongoing studies will allow us to discover the mechanism by which SP-A modulates the effect of IL-4 in aMϕ and determine if SP-A together with IL-4 causes either aMϕ survival or proliferation.
Infection, especially by helminths or bacteria, can cause tissue damage (see the Perspective by Bouchery and Harris). Minutti et al. studied mouse models of helminth infection and fibrosis. They ...expressed surfactant protein A (a member of the complement component C1q family) in the lung, which enhanced interleukin-4 (IL-4)-mediated proliferation and activation of alveolar macrophages. This activation accelerated helminth clearance and reduced lung injury. In the peritoneum, C1q boosted macrophage activation for liver repair after bacterial infection. By a different approach, Bosurgi et al. discovered that after wounding caused by migrating helminths in the lung or during inflammation in the gut of mice, IL-4 and IL-13 act only in the presence of apoptotic cells to promote tissue repair by local macrophages. Science, this issue p. 1076, p. 1072; see also p. 1014 The type 2 immune response controls helminth infection and maintains tissue homeostasis but can lead to allergy and fibrosis if not adequately regulated. We have discovered local tissue-specific amplifiers of type 2-mediated macrophage activation. In the lung, surfactant protein A (SP-A) enhanced interleukin-4 (IL-4)-dependent macrophage proliferation and activation, accelerating parasite clearance and reducing pulmonary injury after infection with a lung-migrating helminth. In the peritoneal cavity and liver, C1q enhancement of type 2 macrophage activation was required for liver repair after bacterial infection, but resulted in fibrosis after peritoneal dialysis. IL-4 drives production of these structurally related defense collagens, SP-A and C1q, and the expression of their receptor, myosin 18A. These findings reveal the existence within different tissues of an amplification system needed for local type 2 responses.
The type 2 immune response controls helminth infection and maintains tissue homeostasis but can lead to allergy and fibrosis when improperly controlled. We reveal the existence of local ...tissue-specific enhancers of type 2 mediated-macrophage activation. In the lung, surfactant protein A (SP-A) enhanced IL-4-dependent proliferation and activation of alveolar macrophages, accelerating parasite clearance and reducing pulmonary injury following infection with a lung-migrating helminth. In the peritoneal cavity and liver, C1q enhancement of type 2 macrophage activation was required for liver repair following bacterial infection, but resulted in tissue fibrosis following peritoneal dialysis. Both SP-A and C1q generated their effects on macrophages via the unconventional myosin18A that acts as a cell surface receptor. We conclude that the structurally related defense collagens SP-A and C1q are tissue-specific factors that act through myosin18A to license local type 2 responses with consequences for parasite control, tissue repair, and fibrosis.
PDF
