An Insight into the Role of IL-10 and Foamy Macrophages in Infectious Diseases

Interleukin-10, a potent immune modulator, inhibits innate and adaptive immune responses like phagocytosis, cytokine induction, and Ag presentation by APC-like macrophages and dendritic cells (1, 2). The signaling pathways that mediate the effects of IL-10 are summarized in Fig. 1. APCs recognize pathogen-associated molecular patterns (PAMPs) and secrete proinflammatory cytokines like TNF-α, IFN-γ, etc., which can in turn be regulated by IL-10 in a feedback loop, thereby inhibiting TLR-dependent APC activation (except TLR3) and downstream TLR signaling pathways (3). An example of this is the “superinduction” of LPS-induced proinflammatory cytokines (TNF-α, IL-1β, and IL-12) in type I IFN signaling-deficient macrophages, which are incapable of producing IL-10 via STAT3 phosphorylation (4). Although IL-10 is crucial for controlling host inflammatory response and preserving immune homeostasis, there are also reports suggesting that IL-10, due to its immune-suppressive function, may accelerate the establishment of infectious diseases (3, 5, 6). When expressed in an untimely manner or when its expression is manipulated by the pathogen, IL-10 may inhibit the host’s protective immune responses by blocking the production of proinflammatory cytokines, like TNF-α, IL-12, etc. In addition, IL-10 can 1) directly act on the APCs and interfere with APC functions, 2) inhibit phagocytosis, and/or 3) restrict microbial killing through limiting the production of reactive oxygen species and nitrogen intermediates (2, 6). This may create a favorable condition for the pathogens to establish an infection. The impact of IL-10 in various infections is summarized in Table I.

Dysregulated lipid metabolism in macrophages gives rise to lipid bodies amassing either triacyl glycerol–, cholesterol-, or phospholipid-rich droplets, resulting in the foamy appearance (34). Foamy macrophage (FM) biogenesis comprises complex coordinated steps of lipid input, metabolism, storage, and mobilization associated with chronic inflammation and infectious diseases (34). FMs have been well documented in several inflammatory metabolic disorders like atherosclerosis, hyperlipidemia, diabetes, insulin resistance, and obesity; cancer (non-small lung cancer, esophageal xanthoma, papillary renal cell carcinoma); and autoimmune conditions like multiple sclerosis, rheumatic diseases, and systemic lupus erythromatosis (reviewed in references 34 and 35). In addition, FMs are a characteristic feature of several intracellular bacterial and parasitic infections like tuberculosis (TB), leprosy, chlamydiasis, salmonellosis, toxoplasmosis, and infection with Klebsiella sp., Trypansoma cruzi, or Chagas’ disease (21, 36–41). Recently, proinflammatory FMs were shown to develop in a model of extracellular Trypansoma carassii infection in zebrafish (42).

IL-10 plays a crucial role in FM generation (summarized in Fig. 2). This is well demonstrated in il10^−/− mice infected with Klebsiella rhinoscleromatis, which had lower frequencies of inflammatory FM known as “Mikulicz cells” and consequently had reduced bacterial burden underpinning 1) the importance of IL-10 in the generation of a foamy phenotype and 2) the fact that FMs serve as sites of bacterial replication and survival (21). IL-10 plays a crucial role in FM generation during TB as well (43). Human monocyte-derived macrophages, when treated with pleural effusion from TB patients, develop a foamy phenotype and acquire immune-suppressive properties like increased expression of IL-10, CD163, and PD-L1 expression and reduced TNF-α secretion (43). However, this phenomenon is not observed upon treatment of monocyte-derived macrophages with IL-10–depleted pleural effusion, underpinning the critical role of IL-10 in the FM differentiation process (43). Additionally, alveolar macrophages as observed in fixed sections of lung biopsies from individuals with untreated TB were found to be foamy in nature, to express M2 markers CD68 and CD163, and to have high levels of surface PD-L1, suggesting that they are immune suppressive (44). FM generation via IL-10 and TLR-mediated mechanisms has been reported in instances of infection by other mycobacteria like Mycobacterium leprae as well (45).

Macrophage differentiation occurs in response to environmental cues like cytokines and microbial PAMPs. Broadly and traditionally, macrophages are classified into classically activated proinflammatory M1 and alternatively activated, anti-inflammatory/regulatory M2 phenotypes (46). M1 macrophages are formed in response to IFN-γ and LPS, whereas M2 macrophages arise in the presence of either IL-4 + IL-13 (M2a) or TLR ligands (M2b) or IL-10 (M2c) (47). Transcriptome analysis has helped in identification of a wide spectrum of macrophage categories each having a unique molecular signature (48). Macrophages are therefore highly heterogenous and possess immense plasticity, and their categories appear to exist as a continuum (reviewed in references 46, 47, 49, and 50). Evidence demonstrates that in atherosclerotic plaques, cancer, and infections like tuberculosis, FMs display anti-inflammatory properties akin to those observed in M2-type macrophages, which are regulatory, have reduced capacity for NO generation, are less microbicidal, and promote tissue repair (51–53). Evidence of the link between M2 macrophages and the foamy phenotype comes from studies in which alternative differentiation of macrophages from diabetic patients, in the presence of IL-4, IL-10, or OVA immune complex, leads to increased FM formation compared with classical differentiation in the presence of IFN-γ (53). The induction of the FM is mediated by expression of scavenger receptor SR-A1 and CD36 and endoplasmic reticulum stress-mediated JNK activation and increased peroxisome proliferator-activated receptor-γ (PPAR-γ) expression (53). However, it is important to point out that in a model of extracellular trypanosome infection of zebrafish, the FMs generated were proinflammatory and contributed to susceptibility to infection (42), highlighting the heterogeneity of FM, which can be dependent among other things on the nature of the pathogen: e.g., intracellular versus extracellular. The M2-like nature of FMs promotes establishment of infection in TB and tumor activity in cancer; however, it has been suggested that M2 FMs in atherosclerotic lesions can be antiartherogenic by countering the proinflammatory M1 macrophages (51).

FM formation during infection is affected by IL-10 as well as pathogen-induced alteration of lipid homeostasis pathways. This review focuses on three major aspects: 1) contribution of IL-10 to pathogenesis and FM formation during various infections, 2) pathways involved in FM formation and how the IL-10–FM axis is exploited by various pathogens to favor intracellular survival, and finally, 3) how the IL-10–FM signaling axis can serve as a target for therapeutic interventions in infectious disease.

Macrophages are flexible and often adapt their functions according to environmental stimuli such as stress, infection, disease, etc. Dysregulated lipid metabolism in macrophages causes the production of lipid droplets or lipid bodies, in which they can accumulate triacylglycerol, cholesterol, enzymes like cyclooxygenases 5- and 15-lypoxygenases leukotriene C4-synthase, phospholipase A₂, MAP kinases, or other phospholipid-rich droplets, resulting in the foamy appearance of the macrophages (34, 54). Such lipid-rich FMs are exploited by pathogens for a variety of purposes. They can serve as nutrient reservoirs, interfere with host signaling cascades, or help in immune escape (36). A classic example of this is Mycobacterium tuberculosis (Mtb), which efficiently uses host cholesterol to meet its carbon and energy requirements with the help of the protein Mce4 (55). Mtb infection increases the expression of microRNA (miR-33) in macrophages suppressing lipid catabolism via autophagy to preserve intracellular lipid stores (56). Within FMs, Mtb uses host lipids for synthesis of its cell wall and survival during dormancy (3). Interestingly, not just Mtb but also other pathogens promote FM generation, leading to clinical manifestations (54). Various factors and signaling cascades involved in FM formation and how various pathogens manipulate IL-10–FM signaling to favor its intracellular survival are described below and summarized in Table II.

In macrophages, the recognition of microbial PAMPs by TLRs triggers signaling events leading to FM formation (summarized in Fig. 2). Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection in C57BL/6 mice induces TLR2-dependent FM formation (57). The same study also demonstrated that the accumulation of lipid bodies in macrophages was directly linked to the presence of PGE₂ and IL-10 (58). M. leprae infection–induced lipid droplet accumulation in mouse peritoneal macrophages was found to be partially dependent on TLR2 and TLR6 (45). Also, culture medium from such infected cells had a high IL-10/IL-12 ratio and could induce a foamy phenotype in uninfected cells (45). M. leprae–infected Schwann cells from lepromatous leprosy patients and have a foamy phenotype dependent on TLR6 but not on TLR2 (58). Additionally, M. leprae–induced lipid droplet biogenesis correlates positively with increased IL-10 and PGE₂ and reduced IL-12 and NO, highlighting the M2-like nature of FM and underpinning the importance of IL-10 in conjunction with TLR signaling in the formation of FM (58). These reports suggest that TLR signaling and PGE₂ and IL-10 expression act in concert during mycobacterial infection to induce lipid droplet biogenesis and FM formation. The TLR involved, however, can vary depending on the infectious organism and the nature of the macrophage.

FMs are associated with conditions of chronic inflammation ranging from noninfectious atherosclerotic plaques to infectious tuberculous granulomas. The biogenesis and nature of the FM is disease dependent; for example, FMs in tuberculosis accumulate triglycerides, as opposed to FMs in atherosclerosis, which are rich in cholesterol (34). Similarly, the origin of FMs in various infection conditions is also heterogenous and can be influenced by several cytokines. In TB, the generation of host proinflammatory responses against an infection like increase in IFN-γ can drive FM formation (66). IFN-γ–induced HIF-1α signaling drives lipid droplet accumulation in Mtb-infected mouse bone marrow–derived macrophages (BMDMs) as well as a mouse model of in vivo Mtb infection (66). Importantly, Mtb cannot use host lipids from droplets that are formed in the presence of IFN-γ, suggesting that this might be a protective host immune response (66). In another example, Chlamydia pneumoniae, an obligate intracellular pathogen known for accelerating atherosclerosis, induces proinflammatory IL-1β through NLRP3 activation, and this in turn inhibits Gpr109a- and ABCA1-mediated cholesterol efflux, leading to accumulation of cholesterol and formation of foamy macrophages, in which C. pneumoniae can survive and persist (61). This is an excellent example of a pathogen exploiting a feedback mechanism to create a survival niche (61). As opposed to the above examples, pathogens like the actinomycete Nocardia brasiliensis induce an immune-suppressive response characterized by expansion of T regulatory cells and elevated TGF-β and IL-10 in infected mice (59). Also, in vitro N. brasiliensis infection in murine BMDMs and bone marrow–derived dendritic cells promotes intracellular accumulation of lipids and development of a foamy phenotype (74). CFSE-labeled BMDMs and bone marrow–derived dendritic cells when transferred into actinomycetoma lesions of infected mice were positive for staining for lipid droplets (60). Even though there is no direct evidence, it can be speculated that the immune-suppressive environment observed during in vivo N. brasiliensis infection might have a role to play in formation of FM (59, 60). It is worth pointing out that as a contrast to observations in murine models of infection in which IFN-γ was found to play a critical role in FM formation, a positive correlation was found between IL-10 levels and pleural CD14⁺ lipid-laden cells from TB patients, which could mean that IL-10 either was secreted by FM or was involved in their generation (17). Cell free TB pleural effusion is capable of inducing FM formation in monocyte-derived macrophages from healthy donors; however, IL-10–depleted pleural effusion lost this capacity, underpinning the role of IL-10 in FM formation (43).

PPARs are transcription factors that maintain homeostasis between lipids, lipoproteins, and glucose metabolism. PPAR-γ binds to fatty acids and eicosanoids and is involved in modulating lipid biogenesis (74). TR4 (testicular receptor 4) functions as a lipid sensor to induce FM partly by regulating oxLDL receptor CD36 (52, 75). PPAR-γ and TR4 contribute to lipid biogenesis by regulating oxLDL receptor CD36, inhibiting phagolysomsome maturation, increasing IL-10, and enhancing alternate polarization of macrophages, eventually leading to the creation of a foamy niche, which enables intracellular Mtb survival (52). Silencing of PPAR-γ and TR4 results in reduced intracellular Mtb survival, IL-10, and arginase activity and increased NO, IL-6, and TNF-α, demonstrating the critical role of the PPAR-γ/ΤΡ4−IL-10 alternate polarization axis in creating a foamy phenotype conducive to Mtb survival (52). TLR2-induced PPAR-γ was found to be important for the formation of lipid bodies during infection with M. bovis BCG (67). C. pneumoniae infection induces FM formation by upregulating ACAT1 (acetyl-CoA acetyltransferase 1) through a PPAR-γ-dependent pathway (69). Examples of other transcription factors involved in FM formation are the microRNA miR-129–regulated Sp2 in Mycobacterium avium–infected macrophages (68) and the liver X receptor (LXR), a cholesterol efflux regulator, in C. pneumoniae infection (70, 76). However, there is no evidence of the involvement of IL-10 in FM formation mediated either by Sp2 or LXR.

The generation of a FM results from breakdown in the balance between the influx and efflux of lipoproteins. Free cholesterol from lipoproteins, endocytosed through cell surface receptors such as CD36, is esterified by the endoplasmic reticulum enzyme ACAT, leading to the formation of lipid droplets inside FMs (77). Evidence for the critical role of ACAT in the formation of lipid droplets comes from studies in which dexamethasone was shown to upregulate ACAT, consequently leading to the formation of FMs (78). Inhibition of ACAT causes accumulation of free cholesterol, leading to the induction of a stress response, resulting in apoptosis (79). Pleural effusion from TB patients was found to be capable of increasing ACAT expression in human monocyte-derived macrophages, leading to an increase in the synthesis of lipid bodies and development of a foamy phenotype in these cells (43). This phenomenon was not observed when IL-10 was depleted from the pleural effusion or when ACAT was inhibited using a specific inhibitor, Sandoz 58-035 (43).

Apolipoprotein E (ApoE), a glycosylated protein, is an important component of triglyceride-rich lipid complexes, which helps in lipoprotein clearance from blood. Apoe^−/− mice, due to defective clearance of very-low-density lipoproteins and chylomicrons, develop atherosclerosis (72, 80). Further, atherosclerosis is accelerated in ApoE and IL-10 double knockout mice, demonstrating that the presence of IL-10 is protective during atherosclerosis (81). Conversely, increase in IL-10 in Apoe^−/− mice during hypoxic conditions coincided with decreased plaque formation (82). Interestingly, Apoe^−/− mice are susceptible to fungal, viral, and bacterial infections, including tuberculosis, due to their inability to mount protective innate and adaptive immune responses (83). Currently, there is no study that directly links IL-10, ApoE and FM during infection. However, evidence from studies on atherosclerosis and the Apoe^−/− mouse model warrants further exploration of this link.

In skin lesion biopsies from disseminated lepromatous leprosy patients, ApoB, the major lipoprotein of oxLDL, colocalizes with CD209⁺CD163⁺ IL-10–secreting foamy macrophages, which are heavily infected with M. leprae as opposed to self-healing, contained tuberculoid lesions (71). This provides evidence that 1) FM are niches for mycobacterial growth and 2) a link exists between the IL-10–secreting M2-like nature and FMs.

IL-10 promotes cholesterol uptake from modified lipoproteins in macrophages and their transformation into FM by increasing the expression of scavenger receptor SR-B1 (CD36) and scavenger receptor A (SR-A) (75). Membrane proteins like fatty acid translocase (FAT/CD36), MSR1/CD204, and macrophage receptor with collagenous domain (Marco) mediate the uptake of LDL into macrophages, which are then metabolized to triacylglycerides, phospholipids, and cholesterol, leading to the development of a foamy phenotype (84, 85). TB pleural effusion increases CD36 expression and foamy phenotype in healthy macrophages in an IL-10–dependent fashion (43). Foamy M. leprae–infected macrophages in lepromatous leprosy lesions show increased accumulation of cholesterol and higher RNA and protein levels of LDL-R, CD36, SRA-1, SR-B1, and LRP-1 (37). These same lesions were also reported by the same authors to have a high IL-10/IL-12 ratio dependent on TLR2 (45), underpinning the nexus between TLR signaling, IL-10, and scavenger receptor expression, leading to accumulation of lipid droplets in infected macrophages.

IL-10 is an immune function modulator that inhibits infection-induced Th1-type cytokines (3). However, in the chronic phase of infection, elevated IL-10 can limit pathology due to exaggerated inflammation (86). Finding the cellular origins of IL-10 in response to infectious diseases may help to develop immune therapeutics to treat infections and their pathogenic sequelae, such as FM favoring pathogen survival and chronicity (6, 87). Several therapeutics that target IL-10 or FM formation have been proposed to prevent intracellular survival of pathogens, leading to chronic infectious conditions (88, 89).

Mtb induces IL-10 in vivo as well as in vitro and the role of IL-10 in TB has been extensively studied (86, 90). Host-directed therapies targeting IL-10 to improve TB prognosis have produced promising results (91, 92). Blocking IL-10 by administering anti–IL-10 receptor 1 (IL-10R1) during Mtb infection in susceptible CBA/J mice reduced bacterial load and increased survival (93). Administration of IL-10R1 along with BCG vaccination resulted in better bacterial control and survival in CBA/J mice upon subsequent Mtb challenge (92). This was associated with presence of higher frequencies of CD4⁺ and CD8⁺ memory cells and reduced Th1/Th17 cytokines (92). Aerosol delivery of peptide inhibitors/analogs targeting either STAT3 or IL-10 receptor α chain (IL-10Ra) in Mtb-infected mice increased NO but decreased arginase activity, ultimately leading to reduced bacillary load (91). Similarly, blockade of IL-10R in Leishmania donovani–infected BALB/c mice resulted in rapid parasite killing, reduced tissue damage and increased responsiveness to antimony chemotherapy (31). Another novel strategy to counteract IL-10 during Mtb infection would be to develop inhibitors that target the 11–15 leucine-rich repeat domains of TLR2, which binds to Mtb proteins like PPE18, leading to elevated IL-10 (90).

In TB, lipid metabolic pathways can also be targeted for host-directed therapy to reduce FM production. Statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA reductase and are commonly used for treating atherosclerosis and hyperlipidemia, could potentially be used for TB treatment (94). Administration of simvastatin reduced the duration of TB chemotherapy in Mtb-infected mice (95), and use of lovastatin in in-vitro M. leprae–infected mice diminished cholesterol colocalization to pathogen-containing phagosomes and decreased intracellular survival of M. leprae (37). Statins, e.g., fluvastatin, simvastatin, and atorvastatin, have been found to inhibit TLR4-mediated inflammatory pathways that lead to lipid droplet biogenesis (96). Known antioxidant resveratrol suppresses superoxide anion and IL-17A levels in C. pneumoniae–infected macrophages and inhibits their conversion to FM (62). This impact of resveratrol is overridden by GW9662, a specific PPAR-γ agonist, suggesting that perhaps resveratrol functions via suppression of PPAR-γ–mediated FM formation pathways (62). GW501516, a PPAR-δ agonist that increases cholesterol efflux (97), and GQ-177, a PPAR-γ partial agonist that reduces atherosclerosis in Ldlr^−/− mice fed a high diabetogenic diet (97), could also be used to correct metabolic imbalances and FM formation in infections. Similarly, synthetic LXR and retinoid X receptor agonists reduced atherosclerosis in Ldlr^−/− and Apoe^−/− mice by promoting cholesterol efflux (98, 99).

The study of FM in infection has provided valuable insights into the signaling events associated with the generation of FM. The formation of FM is deeply linked to the nature of the infecting organism, and the pathways can vary substantially. However, within these intricate processes, IL-10 emerges as a pivotal modulator, influencing immune and metabolic regulatory factors. Therefore, the use of immune-based host-directed therapies, like IL-10 receptor Ab (92, 93), represents the next generation of treatments that are being pursued with great interest for developing effective therapeutics to manage infectious diseases and associated clinical pathology (100).

Given the complexity of the immune response, it is advisable to study the cellular and molecular processes of IL-10 and FM together to get a holistic overview. Considering recent advances, it is evident that by expanding our knowledge on IL-10–mediated FM formation, new (and safer) therapeutic targets could be identified for diseases that are often associated with poor clinical outcome such as TB. This insight is further supported by the extensive studies on FMs and their role in disease pathogenesis, notably in atherosclerosis (101, 102), and could serve as an excellent example of how one can exploit the potential of IL-10 as an immune-therapeutic target against diverse pathogenic challenges. The specific molecular events in IL-10–induced FM formation warrant further investigation for a comprehensive understanding.

The authors have no financial conflicts of interest.

We thank Niteen Pathak for help and suggestions. The figures are created using Biorender.com, an illustration tool, in accordance with its term and services.