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Title: Inflammatory Stimuli Reprogram Macrophage Phagocytosis to Macropinocytosis for the Rapid Elimination of Pathogens}
Article:
Abstract:
Following an infectious challenge, macrophages have to be activated in order to allow efficient clearance of infectious pathogens, but how macrophage activation is coupled to increased clearance remains largely unknown. We here describe that inflammatory stimuli induced the reprogramming of the macrophage endocytic machinery from receptor-mediated phagocytosis to macropinocytosis, allowing the rapid transfer of internalized cargo to lysosomes in a receptor-independent manner. Reprogramming occurred through protein kinase C-mediated phosphorylation of the macrophage protein coronin 1, thereby activating phosphoinositol( PI)-3-kinase activity necessary for macropinocytic uptake. Expression of a phosphomimetic form of coronin 1 was sufficient to induce PI3-kinase activation and macropinocytosis even in the absence of inflammatory stimuli. Together these results suggest a hitherto unknown mechanism to regulate the internalization and degradation of infectious material during inflammation.
Introduction:
Macrophages are the main scavengers responsible for clearance of solutes and particulate material as well as to act as defense cells against invading microbes[1]. The main mechanisms via which macrophages can internalize and clear microbial material occurs through receptor-mediated phagocytosis. This process, making use of different cell surface receptors, including Fc receptors, complement receptors, scavenging receptors as well as several lectin receptors, ensure the uptake of particulate material into phagosomes followed by delivery of the cargo to lysosomes[1]. Under certain conditions phagocytosis is not sufficient for an effective elimination of microbial pathogens. For example, pathogenic mycobacteria, which include the causative agent of tuberculosis, can be internalized via phagocytosis using different receptors, including complement receptor, scavenging receptors as well as lectin receptors such as the mannose receptor and DC-SIGN[2][1]. Once internalized into phagosomes, pathogenic mycobacteria have evolved to withstand lysosomal degradation by effectively blocking phagosome-lysosome fusion thereby surviving within macrophage phagosomes instead of being degraded in lysosomes prior to cytosolic escape[3]–[10]. Also, during an acute infection, the phagocytic capacity of macrophages may become limiting in being able to internalize and destroy sufficient numbers of bacilli in order to curb the infection[11]. Furthermore, the particular receptor involved may modulate the macrophage killing capacity by silencing certain macrophage responses such as the respiratory burst[12]. As an alternative to receptor-mediated phagocytosis, macrophages can also internalize material via macropinocytosis, a non-saturable mode of uptake that allows the internalization of large amounts of cargo independent of any receptor usage[11],[13]–[15]. In several cell types, macropinocytosis can be transiently induced by growth factors as well as certain pathogens such as Salmonella, Shigella or viruses[16],[17]. In macrophages, as well as dendritic cells, where macropinocytosis also occurs constitutively, macropinocytosis allows to efficiently process infectious material as well as activate immune responses[14]. Here, we show that inflammatory stimuli reprogram the macrophage endocytic pathway from receptor-mediated phagocytosis to macropinocytosis, enabling macrophages to internalize large amounts of cargo for direct transfer to lysosomes. We found that upon macrophage activation, serine phosphorylation of the macrophage protein coronin 1 is the key molecular switch that reprograms the macrophage from a phagocytic uptake mode to macropinocytosis. Coronin 1( also known as P57 or TACO, for Tryptophan aspartate containing Coat protein), was originally identified as a survival factor for intracellular residing mycobacteria by blocking the delivery of pathogenic mycobacteria to lysosomes via the activation of the Ca2+/calcineurin pathway[18]–[23]. In resting, non-activated macrophages, coronin 1 is associated with the cell cortex via an interaction( either direct or indirect) with plasma membrane cholesterol[24]. We found that upon cytokine-mediated macrophage activation, coronin 1 was phosphorylated on multiple serine residues by protein kinase C, which induced the relocation of coronin 1 from the cortex to cytoplasmic puncta. Serine phosphorylation of coronin 1 was sufficient to induce phosphoinositol-3-kinase activity thereby switching the internalization mode from receptor-mediated phagocytosis to macropinocytosis. Together these results not only provide a molecular explanation for the mycobactericidal effect of macrophage activating cytokines, but furthermore suggest that macrophage activation reprograms the endocytic machinery through coronin 1 phosphorylation in order to efficiently eliminate infectious cargo.
Discussion:
Importantly, the here demonstrated switch in phagocytic to macropinocytic uptake upon macrophage activation is based on several independent lines of evidence; first, morphological analysis by light and electron microscopy revealed that upon macrophage activation cargo was internalized into spacious vacuoles involving large membrane ruffles, which are hallmarks of macropinocytic as opposed to pseudopod-mediated phagocytic uptake[14],[26]–[29]. Second, upon macrophage activation, internalized cargo strongly colocalized with the early macropinocytic markers SNX1 and SNX 5. Third, we made use of three inhibitors that block macropinocytosis via distinct modes of action; while amiloride blocks macropinocytosis via inhibition of the Na+/H+ exchanger[32], 3-methyl adenosine inhibits macropinocytosis via blocking a specific class of PI-3-kinase and blebbistatin blocks macropinocytosis via the inhibition of nonmuscle myosin II[36],[47] all of which have a crucial role in macropinocytosis. Fourth, analysis by flow cytometry showed that upon macrophage activation, internalization of IgG-coated beads occurred in bulk as opposed to single uptake events in non-activated macrophages. Induction of macropinocytosis through cytokine-mediated macrophage activation is an exquisite strategy from the host immune defense point-of-view to efficiently eradicate pathogenic material; First, this pathway provides a way for the immune system to clear large amounts of extracellular material[11],[48]; second, since some receptor-mediated phagocytic entry pathways result in silencing of inflammatory responses[12], avoiding phagocytosis altogether and instead taking up cargo via macropinocytosis ensures a complete microbial clearance. Third, there is little to none communication between macropinosomes and conventional endosomes[49], and it is conceivable that this strict separation between macropinosomes and the phagocytic/endocytic pathway may help to ensure a rapid and efficient clearance of the pathogens through lysosomal degradation in activated macrophages, thereby preventing extensive exchange of microbes to more hospitable subcellular organelles such as used by several intracellular pathogens including Listeria spp., Brucella spp. and Mycobacterium spp. An intriguing finding in this study is that the macrophage protein that functions as a switch from phagocytosis to macropinocytosis is coronin 1. Coronin 1 was identified in a search for molecules that allow intracellular survival of mycobacteria that are being internalized through phagocytosis via one of the macrophage phagocytic receptors[1],[18],[19], and subsequent work revealed a role for coronin 1 in promoting Ca2+/calcineurin signaling upon mycobacterial infection, but any other role for coronin 1 in macrophages has not been defined[21],[50],[51]. Although other members of the coronin protein family are expressed in macrophages[20], the here described role for coronin 1 in acting as a molecular switch to induce macropinocytosis is clearly a non-redundant function, since in coronin 1-deficient macrophages macropinocytosis was not induced. Why coronin 1 is unable to prevent the delivery of mycobacteria from macropinosomes to lysosomes may lie within the phosphorylation-induced monomerization of coronin 1, which is not anymore capable of protecting the macropinosome residing mycobacteria from lysosomal destruction( SBDG and JP, unpublished). A function for coronin 1 in the activation of macropinocytosis is furthermore consistent with the association of coronin 1 with cholesterol[24],[52]. Cholesterol is an essential component of the macropinocytic pathway in the absence of which macropinocytosis cannot occur[48],[53]. Interestingly, the cholesterol-staining agent filipin colocalized with coronin 1 within the cytoplasmic puncta; whether cholesterol is required for an efficient phosphorylation of coronin 1, or rather needed for coronin 1-mediated activation of PI-3 kinase during macropinocytic uptake remains to be established. Whereas the process of phagocytosis is well characterized, the mechanisms involved in macropinocytic entry are only beginning to become elucidated[13],[15],[17], and which proteins exactly are involved in macropinosome formation is still largely unclear[13]. Importantly, constitutive macropinocytosis is unaltered regardless of the activation state of the macrophages and proceeds even in the absence of coronin 1[21]. Although recent work shows the importance of sorting nexin 5 in macropinocytosis, the precise spatio-temporal pattern of signaling events leading to the induction of macropinocytosis as well as the precise role for sorting nexin 5 in macropinosome formation remains unclear[17],[43]. SNX5 is recruited to the plasma membrane via its phosphoinositide-( PX) binding domain that binds to PI( 3, 4) P2[54]. Therefore, the here reported finding that PI-3 kinase activation was dependent on IFN-γ-mediated coronin 1 phosphorylation is consistent with a model in which serine phosphorylated coronin 1 associates with sorting nexin 5 and is targeted to PI( 3, 4) P2-containing plasma membrane microdomains, after which PI-3 kinase activity is induced to initiate macropinosome formation[55], see also Fig. 9. Importantly, the coronin 1-mediated reorganization of the endocytic pathway occurred independently from IFN-γ-mediated activation of gene expression[56], since even in the absence of macrophage activation, expression of a serine phosphomimetic coronin 1 mutant was sufficient to induce macropinocytosis. Thus, inflammatory stimuli, besides inducing the expression of a cohort of genes that are directly involved in microbial killing[56]–[61], can also modulate entry pathways in order to efficiently transfer infectious cargo to lysosomal organelles. Many of the IFN-γ-induced genes contribute to an effective immune response, including up regulation of genes important for the induction of autophagy[56]–[62]. Notably, the late macropinosome/autophagosomal marker LC3b was recruited at late times following cargo internalization, at a time point when early macropinocytic markers were not anymore associated with bacteria-containing vacuoles( data not shown), clearly indicating that coronin 1-mediated macropinocytosis precedes autophagy upon macrophage activation[56],[58],[62]. Coronin 1 is emerging as a leukocyte-specific regulator of intracellular signaling processes, and has been shown to promote both the viability of intracellular mycobacteria as well as T lymphocytes via the activation of Ca2+-dependent signaling[23]. Interestingly, a rise in intracellular calcium was shown to be required for macropinocytosis to proceed in dendritic cells[63],[64], and it is possible that relocalized coronin 1 is responsible for the Ca2+ rise upon induction of macropinocytosis. In conclusion, the work described here defines coronin 1 phosphorylation as a master switch inducing macropinocytic uptake of cargo upon cytokine activation, thereby coordinating induction of an entry pathway that allows for the macropinocytic engulfment of large amounts of cargo with an up regulation of genes involved in the antibacterial response. It will be interesting to establish whether or not the manipulation of this pathway may be useful in the development of therapies to induce cargo transfer to lysosomes, including the shuttling of pathogenic mycobacteria to lysosomes for rapid elimination.
The main cells that are involved in cleaning up microbial pathogens are macrophages . Upon an infection , macrophages are being recruited to the site of infection by a number of different stimuli . In addition , during an infection , macrophages are also activated by cytokines such as interferon-γ and tumor necrosis factor-α that is released from other immune cells . Such macrophage activation is important to achieve a rapid and efficient clearance of microbial pathogens . In this study , we found that macrophage activation induces uptake through macropinocytosis rather than receptor-mediate phagocytosis . As a consequence , microbial material as well as particles can be internalized far more efficiently; In addition , the internalized cargo is rapidly destroyed within lysosomes . We also provide the mechanisms for the switch from phagocytosis to macropinocytosis , which turned out to be the cytokine-induced phosphorylation of the host protein coronin 1 . Phosphorylated coronin 1 activated the lipid kinase phosphoinositide 3-kinase , which is known to be responsible for the entry of cargo through macropinocytosis . Together these results provide evidence for a hitherto unrecognized mechanisms to regulate the internalization and degradation of infectious material during an infection .