The Role of Ficolins in Lung Injury

Innate immunity is the first line of defense of the human immune system and relies on innate immune cells to protect the body from infection through the recognition of pathogen-associated molecular patterns by pattern recognition molecules (PRMs) [1]. PRMs can be divided into two categories, cell-associated pattern recognition receptors and extracellular soluble pattern recognition receptors; ficolins are part of the latter category and were first identified in 1993 as transforming growth factor-b1-binding proteins in porcine uterine membranes [2]. When ficolins bind to mannose-binding lectin-associated serine proteases (MASPs), they trigger complement activation via the lectin pathway, thereby mediating a range of immune responses [3]. In addition, recent studies have shown that ficolins can influence the extent of lung injury, attenuate or exacerbate lung injury caused by different factors, and play multiple roles in lung disease in a variety of ways. In addition to describing the general properties of ficolin as an innate immune recognition molecule, this review summarizes some of the recently published studies on the effects of ficolins on the progression of lung injury.

To date, three types of ficolin (M-ficolin, L-ficolin [L-FCN], and H-ficolin) and two types of ficolin (ficolin A [FcnA] and ficolin B [FcnB]) have been identified in humans and mice, respectively. Although the genes encoding these ficolins are thought to have evolved independently in different lineages, human M-ficolin or ficolin-1 is a homolog of murine FcnB, L-FCN or ficolin-2 (FCN-2) is closely related to murine FcnA, and mouse H-ficolin is a pseudogene [4]. Among human ficolins, L-FCN and H-ficolin (Hakata antigen) are plasma ficolins. L-FCN is synthesized by hepatocytes, and partial L-FCN is also found in the lungs, adrenal glands, adipose tissue, and prostate, while H-ficolin is synthesized by hepatocytes, alveolar II pneumocytes, and ciliated bronchial cells [3, 5, 6]. M-ficolin is synthesized by neutrophils, monocytes, and bone marrow and is found in secretory granules in the cytoplasm of pulmonary neutrophils, monocytes, and alveolar type II epithelial cells [3]. Related studies have shown that M-ficolin is an acute protein that is temporarily stored in leukocyte secretory granules [7]. Compared to macrophages, monocytes and neutrophils have a larger reservoir of M-ficolin [8, 9] (Table 1). FcnA in mice is functionally equivalent to L-FCN in humans, is present in serum, and is expressed in splenic macrophages and Kupffer cells. FcnB, which is functionally equivalent to M-ficolin, is present on cell membranes and in the granules of neutrophils and monocytes but not in serum [10, 11]. In addition, FcnA and FcnB have been found to be expressed in bone marrow, peripheral blood, and lung and spleen leukocytes, and further analysis has shown that macrophages and neutrophils are the major sources of FcnA and FcnB, with T cells and B cells also expressing small amounts of FcnB [12] (Table 1). Ficolins consist of an N-terminal collagen-like domain and a C-terminal fibrinogen-like domain and form trimer-based multimers via disulfide bonds between the N-termini of the trimers (Fig. 1a). The protomers of human ficolins have A, B, and P structural domains (Fig.1b) [13]. The ligand binding site existed in P domain was near the Ca2+ binding site on the external part, which was named for S1 [14]. Based on the X-ray crystal structure of the recognition domains of the ficolins, it was revealed that L-FCN contains four carbohydrate recognition domain (CRD) sites (termed S1–S4), whereas only one site was detected in M-ficolin and H-ficolin (Fig.1b) [15]. The presence of multiple binding sites in L-FCN may explain its extensive binding capacity. Binding to acetylated, in particular N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine, is a common characteristic shared among the ficolins. Ficolins generally recognize polysaccharides on microbial pathogens (not necessarily of carbohydrate nature), including N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, and d-galactose. It has been demonstrated that all identified ficolins are capable of recognizing GlcNAc. Terminal GlcNAc residues are widely present on a wide range of pathogens, but not in human cells [16]. Moreover, ficolins have also been observed to recognize specific microbial patterns such as sialic acid, lipopolysaccharides (LPSs), bacterial peptidoglycan, and fungal 1,3-β-D-glucan [17]. The major difference between FcnA and M-ficolin is that it has ten exons, and the two extra exons translated Gly-X-Y repeats and an additional neck sequence, respectively. MASP binding site on FcnA is a conserved motif which is lysine residue in the X position of the Gly-X-Y collagen repeat [18]. Slightly different from M-ficolin, the ligands of FcnA are a trisaccharide only containing a terminal α1-6-linked GlcNAc residue and a few sialated ligands [19]. FcnB shared 61% amino acid sequence semblance with FcnA, but FcnB is 2,502 Da smaller than FcnA. FcnB exclusively bounding to sialyated structure, such as α2-3, α2-8, and β2-6 linkages, has a more broaden recognition than FcnA [19]. Based on this structure, ficolins recognize a variety of pathogen-associated molecular patterns and trigger activation of the immune system (activation of the lectin complement pathway; modulation of phagocytosis; stimulation of inflammatory cytokine secretion; modulation of inflammatory cell function), thereby limiting infection and orchestrating adaptive immune responses (Table 1).

Lung injury is one of the most common clinical respiratory diseases and is caused by the exposure of lung tissue to a variety of stimuli, including hypoxia, ischemia-reperfusion, and xenobiotics [20]. Lung injury is characterized by disruption of the pulmonary endothelial and epithelial cell barriers by uncontrolled inflammation [21]. Lung injury can be caused by a variety of factors, with infectious diseases and interstitial lung disease (ILD) being the most common causes, as well as rheumatic immune disorders, lung transplantation (LTx), and malignancy. Ficolins play a role in all of these processes by recognizing pathogens, regulating immune cells, and activating the complement cascade and can be either a friend or foe to the human body.

Previous studies on ficolins have shown that both human and murine ficolins can bind to a wide range of different pathogenic microorganisms and play a protective role in the body’s immunity [22‒26]. L-FCN in humans and FcnA in mice are the major players in bacterial infectious lung injury (Fig. 2a). L-FCN is one of the major PRMs in human serum and plays an important role in respiratory immunity [27]. Serum levels of L-FCN vary between individuals and are mainly genetically determined. Single-nucleotide polymorphisms in the promoter region and exon 8 have a significant effect on serum levels [28]. Inadequate levels of L-FCN suggest the presence of chronic or infectious lung disease [29]. LPS, also known as endotoxin, is a major component of the cell wall of gram-negative bacilli and is widely used to induce aseptic models of inflammation [30]. Local activation of ficolin plays a key role in host defense against pulmonary LPS challenge [31, 32]. This effect was demonstrated in an experimental study by Wu Xu et al. [12] in 2020, who reported that FcnA, which is secreted by macrophages and neutrophils as a result of local inflammation and protects against LPS-induced mild acute lung injury by activating complement. The role of L-FCNs, which bind to Streptococcus pneumoniae to activate the lectin pathway of complement, in community-acquired pneumonia has been the focus of attention [22, 33, 34]. In addition, FcnA in mouse serum exerts an anti-inflammatory effect by interfering with the interaction between endotoxin and TLR4 on mast cells to attenuate the tissue-damaging effects of the LPS pathogen [35]. A research team previously reported a high incidence of L-FCN insufficiency in patients with severe community-acquired pneumonia [36] and increased mortality in mice lacking FcnA, the mouse homolog of L-FCN, in a S. pneumoniae model [23, 24]. S. pneumoniae releases relatively high levels of pneumolysin (PLY), which binds to eukaryotic cell membranes and causes cell lysis leading to lung tissue damage, shortly after infection, and complement activation and direct combination of PLY with L-FCN (PLY does not bind to mouse serum ficolins) protect the body; complement activation is mediated via activation of the classical pathway by the nonspecific combination of PLY with nonimmune IgG3/IgM and activation of the lectin pathway by the combination of L-FCN with S. pneumoniae [37‒41]. However, in opportunistic pathogenic Pseudomonas aeruginosa infections, there is reciprocal crosstalk between L-FCN and CRP; in healthy human serum, only CRP binds to bacteria, resulting in low complement activity. Under infectious inflammatory conditions (acidosis, hypocalcemia, and high CRP levels), CRP forms a complex with L-FCN and recruits L-FCN to the bacterial surface. This interaction gives rise to two novel complement amplification mechanisms: PC (a chemical moiety of lipoteichoic acid)→CRP: L-FCN→MASP-2→C4→C3→membrane attack complex (MAC) and GlcNAc (a chemical moiety of LPS)→L-FCN:CRP→C1q→C4→C2→C3→MAC. This consequently results in the upregulation of the formation of the MAC, which exhibits antibacterial activity against invading pathogens [42]. Interestingly, two recent studies have shown another “face” of ficolins; one study investigated the interaction between FcnA and the gut microbiota to study sepsis-induced lung injury. FcnA exacerbated LPS-induced acute lung injury by activating the S100A4/STAT3 signaling pathway, in which the gut microbiota (e.g., Akkermansia) plays an important role [43]. Another study investigated the role of FcnA, pyroptosis, and NETs in LPS-induced lung injury. This study showed that in a mouse model of LPS-induced lung injury, the FcnA protein activates caspase-1, which promotes the cleavage of GSDMD to GSDMD-N, thereby inducing neutrophil pyroptosis and exacerbating LPS-induced lung injury by regulating NET formation [44]. In summary, it appears that ficolins act as “ double agents ” in organismal infections, protecting in some cases and exacerbating inflammation through signaling pathways in others, and whether they are protective or not may depend on the course and severity of the inflammation.

Aspergillus is a common mold pathogen that predisposes immunocompromised patients to disease. During growth, the cell wall polysaccharides β-glucan and chitin are progressively exposed and can be immunodetected by a PRM, the β-glucan-binding receptor (dectin-1) [45], which triggers a series of responses to facilitate clearance of the pathogen (Fig. 2b). A previous study revealed that M-ficolin is present in human Aspergillus globules and binds to Aspergillus fumigatus in a calcium-dependent manner; M-ficolin also binds to Aspergillus and mediates complement activation. The M-ficolin-Aspergillus conjugate increased IL-8 secretion by A549 cells, which are a widely used model of type II alveolar epithelial cells, suggesting that M-ficolin mediates the initiation of inflammation and promotes neutrophil recruitment [46]. In 2013, Stefan and colleagues reported that FcnA could play an important role in innate defense against Aspergillus by immobilizing the fungus through the modulation of mycelial sporulation, the enhancement of fungal adhesion to epithelial cells, and the modulation of inflammation [47]. In their 2016 study, they found that FcnA was able to bind to and lyse A. fumigatus, the pathogen that causes invasive aspergillosis, and the modulatory effects of FcnA also reduced the production of IL-8, IL-1β, IL-6, IL-10, and TNF-α by monocyte-derived macrophages, as well as the production of IL-1β, IL-6, and TNF-α by neutrophils, up to 24 h after infection [48]. In 2015, they reported that L-FCN has immunomodulatory properties (macrophages are involved in the phagocytosis and killing of early hyphal spores, while neutrophils are recruited to help at a later stage when extracellular killing mechanisms are indispensable [49]) that enhance the ability of macrophages and neutrophils to kill the fungus and induce an anti-inflammatory cytokine profile after infection [6]. In another study, Stefan and colleagues reported that H-ficolin recognizes A. fumigatus conidia in a calcium-dependent and pH-independent manner and mediates activation of the lectin pathway (H-ficolin binding was significantly reduced when calcium was removed from the buffer and the excess calcium was chelated by ethylene glycol tetra acetic acid; maximum binding was observed under acidic [pH 5.7] conditions) [50]. Although H-ficolin bound significantly to A. fumigatus conidia, its binding activity was much lower than that of serum L-FCN or its murine homolog FcnA, probably because H-ficolin has a much lower affinity for GlcNAc than L-FCN [50]. The above studies revealed that FcnA, L-FCN, H-ficolin, and M-ficolin all bind to Aspergillus to modulate IL-8 secretion from A549 cells [6, 46, 47, 50]. In conclusion, the ficolins previously mentioned contribute to the clearance of Aspergillus conidia from the lungs and modulate cytokine secretion, as well as inflammatory cell recruitment, enhancing host’s defense against Aspergillus and exerting a protective effect on the host.

Cryptococcus neoformans (C. neoformans) is an opportunistic pathogen that causes potentially life-threatening fungal infections in immunocompromised patients (e.g., patients with HIV or leukemia or posttransplant patients) [51, 52]. Ficolins, as soluble PRMs, were demonstrated to play a crucial role in the body’s early resistance to infection by C. neoformans in an in vitro study. The polysaccharide capsule of C. neoformans is a major virulence factor that is composed of a glucuronoxylomannan containing α-1,3-mannose. In addition to recognizing the polysaccharide capsule and binding to C. neoformans to promote the phagocytosis of C. neoformans by A549 cells, FcnA also opsonized acapsular C. neoformans, which significantly enhanced the uptake of yeast by A549 cells; under acidic conditions, the binding affinity of the FcnA pair for C. neoformans increased, suggesting that ficolins may contribute to the early recognition and removal of yeast before it escapes and assembles the protective capsule [53] (Fig. 2c). In C. neoformans infections, FcnA promotes phagocytosis of pathogenic bacteria by immune cells and has the ability to recognize and clear pathogenic bacteria early under acidic conditions.

Tuberculosis (TB) is a major global health problem, with one-third of the world’s population infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB. Although little research has investigated the role and mechanism of ficolins in vitro or in vivo during Mtb infection, some researchers have genetically proposed that the −557A>G, −64A>C, and +6424G>T SNPs in the L-FCN genes are associated with TB and may protect against TB [54]. Mannose-capped-lipoarabinomannan, a glycolipid ligand on the surface of Mtb H37Rv, is an essential component of the Mtb cell wall with immunomodulatory functions that is recognized by intrinsic and adaptive cells [55]. In a combined in vivo and ex vivo study, researchers found that L-FCN can activate the lectin pathway and prevent Mtb H37Rv from targeting alveolar epithelial cells, by using its fibrinogen-like domain to recognize mannose-capped-lipoarabinomannan and then binding to Mtb H37Rv [55]. In addition, L-FCN clears Mtb H37Rv in vivo by activating the phosphorylation of JNK and stimulating the secretion of IFNγ, IL-17, IL-6, TNF-α, and NO by macrophages [55] (Fig. 2d). In conclusion, L-FCN can prevent Mtb H37Rv from targeting alveolar epithelial cells, initiating the lectin pathway and stimulating the production of inflammatory factors that can protect the organism.

Human L-FCN binds to the HA and NA glycoproteins of influenza A viruses (IAVs) and neutralizes viral infection and multiplication [25]. An in vitro study reported that the presence of ficolins in porcine plasma reduced the cytopathological effects and replication of porcine reproductive and respiratory syndrome virus in a GlcNAc-dependent manner [56]. L-FCN has also been reported to directly inhibit IAV entry; L-FCN promotes complement-mediated lysis of IAV particles and infected cells [25] (Fig. 2e). H-ficolin purified from human serum and bronchoalveolar lavage fluid binds to IAV, thereby blocking viral infectivity by inhibiting hemagglutinating activity and viral aggregation, as well as directly blocking complement activation. Individuals with deficient or reduced levels of H-ficolin may also be more susceptible to IAV infection [57‒59]. However, complement activation is not always good for the organism. In a previous report, a mouse model of severe pH1N1 infection showed that FcnA-mediated complement hyperactivation increased the recruitment of inflammatory cells as well as the production of proinflammatory cytokines and chemokines (e.g., TNFα, IL-6, IFNα, IFNβ, IFNγ, G-CSF, CCL2, CCL5, and CXCL1), which exacerbated the proinflammatory response of the pulmonary system, leading to immunopathological damage to the pulmonary system that was not related to viral replication [60] (Fig. 2e). In viral infections, the balance of benefits and harms of ficolin to the host is highly dependent on the viral load; in mild infections (low loads), it outweighs the harms, but in severe infections (high loads) it mediates overactivation of complement, leading to a host inflammatory storm that can exacerbate lung injury. However, the limits of this equilibrium are difficult to identify and experiments need to be designed to continue the exploration.

ILD is a diverse family of diffuse parenchymal lung diseases of known and unknown origin that can be divided into four categories: known associated ILDs (e.g., drugs, connective tissue diseases), granulomatous ILDs (e.g., sarcoidosis), idiopathic interstitial pneumonias, and rare ILDs [61]. Evidence suggests that ficolins play an important role in ILDs (Fig. 3a). Some researchers have shown that pattern recognition receptors of the lectin complement pathway are involved in pulmonary nodularity at the local level and that the levels of L-FCN and H-ficolin are significantly elevated in the lungs of these patients [62]. S. pneumoniae has been found to be abundant in patients with progressive IPF, and in animal models, S. pneumoniae promotes lung fibrosis through PLY-mediated destruction of lung epithelial cells [63]. In addition, high L-FCN plasma levels were protective in patients with IPF [64]. Interestingly, however, in a study using mouse models, bleomycin (BLM)-induced exosomal transport of FcnB by alveolar macrophages accelerated autophagy and iron mutation and exacerbated lung epithelial cell injury by activating the cGAS-STING pathway. In addition, FcnB promotes the process by which BLM leads to the transformation of fibroblasts into myofibroblasts, which produce extracellular matrix-induced collagen fiber deposition and thus exacerbate lung fibrosis injury [65]. Serum H-ficolin levels have been studied in systemic sclerosis-associated interstitial lung disease (SSc-ILD), and it was found that the decrease in serum concentration of H-ficolin was inversely proportional to the grinding glass opacity score, and that the decrease in serum concentration of H-ficolin suggests a dysfunction of the innate immune system of the lungs and results in the accumulation of uncleared apoptotic cells, which are associated with the progression of SSc-ILD, and therefore, H-ficolin may be used as a biomarker for active SSc-ILD [66]. Furthermore, two studies also revealed that high L-FCN levels were correlated with SSc-ILD [67, 68]. L-FCN and H-ficolin, with their elevated concentrations, play a protective role in IPF, SSc-ILD, and pulmonary nodular disease, whereas FcnB exacerbates lung fibrosis damage in BLM-associated ILD. This correlates with the specificity of the function of the different ficolin species.

Ischemia-reperfusion injury (IRI) after LTx activates the complement system, and primary graft dysfunction (PGD), which is mainly induced by IRI, is a form of acute lung injury that affects patient survival after LTx [69, 70]. A study of local complement activation and PGD after LTx suggested that increased airway protein accumulation after IRI may trigger local complement cascade activation in the lung via the lectin pathway, with MBL being a significant marker of lectin pathway activation in patients with PGD and elevated levels of H-ficolin in BALF fluid being highly correlated with markers of complement activation [71] (Fig. 3b). Elevated levels of H-ficolin are associated with complement activation, and its elevation is also a predictor of PGD.

Tumor-related lung injury is mainly characterized by tumor-induced obstructive lung atelectasis, alveolar instability, and susceptibility to coinfection. The effect of ficolins on tumors may indirectly affect tumor-induced lung injury. In 2021, Haeyeon and her group first identified H-ficolin as an oncogene in lung adenocarcinoma, and the expression of H-ficolin in lung adenocarcinoma tissues was lower than that in normal tissues. Interestingly, although H-ficolin is a secreted protein, their study revealed that secretion of the H-ficolin protein into the extracellular matrix does not regulate the cell cycle or apoptotic process but rather acts as a tumor suppressor by inducing endoplasmic reticulum (ER) stress through the induction of H-ficolin inside the ER, whereas the ER stress response leads to cell death [72] (Fig. 3c). Some researchers have suggested that both dysfunction and overactivity of the lectin pathway may be involved in carcinogenesis and disease progression, the former due to the inadequate clearance of abnormal cells and the latter due to damage caused by inflammatory imbalances in the body [73]. There are two sides to the effect of ficolins on tumors: for the tumor itself, H-ficolin has a tumor-suppressing effect, which is beneficial, but for the systemic environment of the body, low levels of H-ficolin can cause increased lung susceptibility.

Respiratory distress syndrome (RDS) in premature infants is a complex disorder associated with inadequate production of pulmonary surfactant by the immature lung, leading to atelectasis, hypoxia, and acidosis [74], the development of which is likely to be associated with impaired clearance of fetal lung fluid [75]; there is also evidence of genetic susceptibility to RDS in preterm infants [76]. A recent study showed that low levels of L-FCN in cord blood serum are associated with RDS in preterm infants. In this study, cord blood samples were collected from 546 Polish preterm infants, and L-FCN concentrations were measured using the sandwich TRIFMA method. Umbilical cord blood serum L-FCN concentrations were positively correlated with Apgar scores, and in neonates born with GA <33, ficolin-2 concentrations could be used to discriminate between babies with and without RDS and were effective at distinguishing babies with mild RDS from those with severe RDS [77] (Fig. 3d). Low serum levels of L-FCN in premature infants susceptible to RDS may be congenital or the result of excessive depletion of the disturbed intra-alveolar environment. For M-ficolin, a prospective study reported that the M-ficolin A/A genotype at position −144 was associated with increased serum levels of M-ficolin, which mediated a hyperinflammatory response leading to adverse effects in Egyptian children under 5 years of age with pneumonia [78] (Fig. 3d). High levels of M-ficolin can mediate excessive complement activation and an excessive inflammatory response, which is detrimental to children with pneumonia.

According to the above data, ficolins play different roles at different stages in the development of diseases; their activation helps the organism defend itself against microbial invasion in the early stages of lung injury, whereas their overactivation exacerbates damage to the organism in the later stages of severe lung injury. Disease-specific complement interventions are therefore essential. Lung injury from all causes has long attracted the attention of the respiratory and critical care medicine community due to the high mortality and morbidity and limited treatment options. New treatments are waiting to be discovered, and ficolin-related complement therapy may be one such opportunity.

The authors declare that they have no conflicts of interest.

This study was supported by National Natural Science Foundation of China, China (82200017); the Science and Technology Innovation Program of Hunan Province, China (2023RC3198); Research Foundation of the Second Affiliated Hospital of University of South China for Young Outstanding Talents (2023G01); and Project funded by Health Commission of Hunan Province, China (20230079, 20201922).

Meiyun Zhao contributed to conceptualization, investigation, validation, and writing of the original draft. Xu Wu and Xiaowu Tan contributed to funding acquisition, project administration, supervision, and review. All authors contributed to the article and approved the submitted version.