The complement system is a fundamental part of innate immunity, the main function of which is to discriminate self and non-self structures in order to keep immune homeostasis. Recognition of foreign particles and modified host cells by complement results in its activation via the classical, lectin or alternative pathways, leading to the cleavage of the central component C3 into C3a and C3b. C3 is the major effector of the complement cascade, causing inflammation by releasing the anaphylatoxin C3a or clearance of the target structures by opsonization with C3b and allowing for further propagation of the cascade, potentially leading to target cell lysis (1). Although complement activation is necessary to eliminate altered self and foreign structures, its over-stimulation might result in attack on the host itself and cause tissue injury. Hence a strict control of complement activation is necessary to maintain immune balance and preserve host integrity (2).

Factor H (FH) is the major regulator of the alternative pathway (AP) of complement by acting as a cofactor for Factor I mediated cleavage of C3b and accelerating the decay of AP C3 and C5 convertases either in solution or when bound to host surfaces (3). FH consists of 20 short consensus repeats (SCR): the N-terminal SCR1-4 domains bind to C3b and exert complement regulatory activity, while the C-terminal SCR19-20 domains ensure binding to host surfaces via recognizing highly sialylated molecules (4, 5). In addition, FH can recognize other polyanionic molecules, like proteoglycans, glycosaminoglycans and other components of the extracellular matrix (6–9), extending the FH mediated protection also to tissues and extracellular spaces. Moreover, to exploit the potential of FH as a therapeutic agent, an artificial mini-FH was generated that contains the regulatory (SCR1-4) and ligand binding (SCR19-20) domains of FH (10, 11). Besides acting as a complement regulator, FH also exerts non-canonical functions via binding to various immune cells, and modulating their activation and function (12). Among those are monocytes and neutrophils, two key components of innate immunity which, similar to complement proteins, circulate in the blood to detect potentially dangerous structures and regulate inflammatory processes (13).

Binding of FH to neutrophils has been reported to modulate a wide range of their functions, among them phagocytosis, ROS production and release of antimicrobial peptides, thus FH may directly influence the ability of the cells to kill pathogens (14–16). FH exerts the vast majority of these effects in immobilized form, serving as an adhesion ligand for the cells, however in soluble form, it is mainly ineffective. Indeed, the increased adherence observed on FH covered surfaces induces rapid polarization of neutrophils, elevation in intracellular Ca²⁺ levels followed by rapid spreading on FH-coated surfaces (14, 16). Similarly, immobilized FH was shown to enhance the release of IL-8 (16), secretion of the antimicrobial peptide lactoferrin and release of hydrogen-peroxide (14), as well as the uptake of Streptococcus pneumoniae (17). Additionally, FH itself also promotes neutrophil migration (16). Besides phagocytosis and intracellular killing, neutrophils can trap and eliminate microorganisms by releasing neutrophil extracellular traps (NETs) (18–20). NETs induce microbial killing by histones and granule-derived antimicrobial proteins, such as myeloperoxidase (MPO), neutrophil elastase and matrix metalloproteinases that associate with these chromatin fibers (20–23). In contrast to the enhanced activation and cytokine secretion, interestingly, the release of NETs by both phorbol-myristate acetate (PMA) or fibronectin plus β-glucan-stimulated neutrophils is decreased in the presence of immobilized FH (16). Similarly to neutrophils, FH was shown to influence a wide plethora of monocyte and macrophage functions too, such as chemotaxis (24), stimulation of respiratory burst (25) and cytokine secretion (26), mainly via the complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) (14, 27). Later, FH binding to Mycobacterium bovis and Candida albicans has been shown to play a role in the evasion of the innate immune system and in parallel, to enhance the pro-inflammatory (IL-1β, IL-6, TNFα) cytokine response of macrophages (27, 28). Monocytes can also produce extracellular traps (MoETs), mainly to enhance killing of extracellular pathogens (29–31). It was shown that FH could bind to NET and MoET directly (29, 32) and bound FH decreases the secretion of the inflammatory cytokine IL-1β in monocytes (29). These data suggest that FH can exert both pro- and anti-inflammatory activities on monocytes and neutrophils, allowing fine-tuning of cellular functions for the regulation of inflammation.

FH belongs to a protein family that also includes the FH splice variant FH-like protein 1 (FHL-1) and five FH-related proteins (FHRs), FHR-1 to FHR-5 (33–35). Similar to FH, they are also exclusively composed of SCRs. Although the complement regulatory region SCR1-4 of FH is not conserved in the FHRs, their C-terminal domains show various degree of sequence identity with C-terminal SCRs of FH. This region represents the major cell surface binding- and C3b recognition part of FH, suggesting that the main function of FHRs is to compete with FH for surface binding and thus modulate complement activation (33, 36). Indeed, both FHR-1 and FHR-5 are competitive inhibitors of FH on different ligands such as C3b, C-reactive protein, pentraxin 3, DNA and extracellular matrix components (37–41). The FH/FHR encoding gene cluster is prone to genetic rearrangements, which are associated with distinct human disorders such as atypical hemolytic uremic syndrome, C3 glomerulopathy, age-related macular degeneration and other autoimmune/inflammatory diseases (33, 42, 43). Due to their role in various diseases, the interest in the FHR molecules has increased, both on the genetic and functional level. Despite the described role of FH in modulating neutrophil and monocyte responses, only a few data are available how FHRs influence the function of these cells. For example, FHR-1 was shown to enhance the adhesion of neutrophils to Candida albicans, causing increased lactoferrin and ROS generation (15), suggesting an FHR-1 mediated regulation of inflammation. Recently, FHR-1 was shown to activate the NLRP3 inflammasome in monocytes when bound to necrotic cells (44).

Considering that both the FHRs and neutrophils and monocytes are key players of inflammatory diseases, we aimed to investigate whether similar to FH, the related proteins FHL-1, FHR-1 and FHR-5, as well as the recently developed mini-FH, a potential complement therapeutic, are able to modulate neutrophil and monocyte functions.

Besides the canonical roles of FH family proteins in the modulation of complement activation, their non-canonical roles as modulators of cellular functions were also observed, but poorly examined yet (12, 33). Among blood cells, neutrophil granulocytes and monocytes were the most extensively studied in this regard as they are essential players in inflammation and innate immunity and they also influence the adaptive immune response. FH binding to neutrophils was first demonstrated in 1993 (58), and later it was shown to be mediated via CR3 (14–17) and to a lesser extent via CR4; the domains within FH responsible for this complement receptor binding were identified as SCR7 and SCR19-20 (15). These SCRs of FH show 100% amino acid sequence identity with the corresponding SCRs of FHL-1 (SCR7) or mini-FH (SCR19-20) (10, 33) ( Figure 8 ). Therefore, FHL-1 and mini-FH have the potential to bind to the cell surface of monocytes and neutrophils similar to FH, which we confirmed by flow cytometry using recombinant proteins ( Figure 1 ). FHR-1 binding to neutrophils was also shown before and is explained by its C-terminal domains that are highly similar to FH SCR19-20; FHR-1 binding could be inhibited by anti-CD11b and anti-CD18 specific mAbs but not by anti-CD11c (15). Moreover, binding of recombinant FHR-5, with less sequence similarity to FH, to the cells was also detected ( Figure 1 ). These results are in agreement with our previous study where neutrophils were incubated with NHS and, in addition to FH, the binding of serum-derived FHL-1, FHR-1 and FHR-5 could be detected by western blot analysis using the cell lysates (15). The binding of FH to neutrophils could be inhibited by FHR-1 but not by FHR-5, further supporting the role of shared binding sites including CR3 for FH and FHR-1 on neutrophils ( Figure 2 ). The relatively high amount of FHR-1 needed for significant inhibition of FH binding suggests that the binding of FH is stronger, e.g. because of the presence of at least two binding sites in FH versus a single site in FHR-1, and is also explained in part by the binding of FH to CR4, as well.

In addition to binding to neutrophil granulocytes, FH, FHR-1 and FHL-1 were described to influence certain cellular functions, but these proteins were studied only in certain aspects (14–17). Here, we studied all these proteins in the same assays and included FHR-5 as well as the artificial construct mini-FH ( Table 1 ). The similar functional effect of mini-FH on the cells compared with FH can be explained by the shared SCR19-20 that are involved in FH binding to CR3 on neutrophils. FHR-5, despite of its strong binding capacity to both neutrophils and monocytes, had no effect on the investigated cell functions except for the TNFα response when applied in combination with LPS, clearly indicating marked functional differences among the otherwise related members of the FH protein family ( Figure 8 ).

Previously, we showed that neither soluble nor immobilized FH alone affected NET production. However, when NET formation was induced with PMA, immobilized but not soluble FH could significantly decrease the amount of extracellular DNA (16). In this study, FHR-1 but not the other FH family proteins or mini-FH had similar inhibitory effect on PMA-induced NET release as did FH ( Figure 4 ). Prolonged or uncontrolled NET formation may enhance the development of inflammation or autoimmune diseases by providing autoantigens, such as DNA or histones. By inhibiting NET production, FH and FHR-1 may limit such undesirable reactions.

During inflammation caused by pathogens or other damage signals, extravasation of neutrophils and monocytes is essential. This is a multistep process including sequential rolling, firm adhesion/spreading and trans-endothelial migration (57, 59). Previous data suggested an adhesive function for FH in the case of neutrophils that is mediated via the complement receptor CR3 (14, 15). In addition, immobilized FH was shown to increase neutrophil spreading through the CR3 receptor, since in our previous study we found that specific antibodies against CD11b could inhibit this process (16), and to support the migration of both neutrophils and monocytes (15, 16, 24). Our present study confirmed the previously described increased neutrophil spreading on FH-covered surface ( Figure 5A ) and the same effect was observed on monocytes ( Figure 5B ). Immobilized FHL-1 and mini-FH had the same effect on both cell types ( Figure 5 ). Interestingly, FHR-1 increased the spreading only in the case of neutrophils which suggests a different regulation process for the two cell types, and may be explained by the differential receptor expression (e.g., CR3 and CR4) on monocytes and neutrophils.

While all of the analyzed FH family proteins and mini-FH showed clear binding to monocytes and neutrophils, they also enhanced the attachment/spreading of the cells and influenced cytokine production, except for FHR-5. Since both cell types are key players in the initiation phase of the immune response, their cytokine production is essential during fighting against infections or can be an important factor in the pathogenesis of inflammatory diseases. We found significant increase in proinflammatory IL-1β secretion of monocytes cultured on mini-FH and slight increase when they were cultivated on FH, FHR-1 and FHL-1 coated wells. Similarly, TNF-α production by monocytes was significantly elevated on wells coated with FH and slightly on those with mini-FH. In the case of neutrophils, IL-8 production was monitored and immobilized FH was shown to enhance the secretion of IL-8 significantly, while FHR-1 and mini-FH induced only a slight increase ( Figure 6 ). The direct role of FH family members in the cytokine production of different immune cells is not clear yet, since controversial findings have been described. Enhanced proinflammatory IL-8 and IL-1β production upon FH treatment was reported, however in most cases FH was applied in combination with other stimuli such as NET-derived DNA or Candida albicans (27, 28). Other studies support an anti-inflammatory role for FH, but again cells were treated with FH plus NET derived DNA, malondialdehyde acetaldehyde BSA or LPS and R848 as TLR ligands (29, 60, 61). We also tested the effect of the different FH family proteins and mini-FH on the cytokine production of LPS stimulated cells. In the case of TNF-α, FH, FHL-1 and mini-FH increased, whereas the FHR proteins FHR-1 and FHR-5 slightly decreased the level of this cytokine. In addition, FHL-1 and mini-FH slightly increased the secretion of IL-10. These results showing that the FH family proteins influence the LPS-triggered TNFα and IL-10 responses of monocytes may suggest their potential involvement in the regulation of inflammatory processes during infection. Interestingly, a recent study also found that mini-FH modulates complement-dependent IL-6 and IL-10 secretion by peripheral mononuclear cells in the presence of autologous serum; IL-10 release was increased by mini-FH when the cells were stimulated with LPS (62). When the effect of the FH family proteins was analyzed in isolated form, FH and FHR-1 treatment induced elevated IL-1β and IL-8 secretion (16, 26, 44) ( Figures 6 and 7 , Supplementary Figure 2 ). In our assays we applied immobilized FH family proteins, and in most other studies FH family proteins influenced cell functions only if they were immobilized on a surface of either a cell culture dish or yeast/bacteria particles. This supports the notion, that FH family members act as circulating molecular sentinels, that continuously monitor for altered structures or invading pathogens to which they can attach and then, in addition to modulating complement activation, they may bind to immune cells and modulate their function.

Soluble FH was found to be a direct modulator of monocyte differentiation since it can induce the differentiation of CD14⁺ human monocytes into immunosuppressive macrophages; this FH effect was inhibited in the presence of autologous plasma, suggesting that this function likely occurs only in tissues (63). Moreover, soluble FH can also generate an anti-inflammatory and tolerogenic state in monocyte-derived dendritic cells (61). However, in these experiments applying several days of co-incubation with FH, the possibility that the added FH may eventually coats the plate surface and thus also acts in an immobilized form, cannot be ruled out. Integrin receptors can sense differences between immobilized and soluble ligands (64, 65), which may explain why we observed an elevated, although not significant IL-1β production upon LPS stimulation on FH coat, while Smolag et al. found a significantly decreased production upon LPS stimulation in FH-treated monocytes (63).

In our experiments we used purified proteins to compare differences in direct cellular functions without adding any other complement component or serum, but it has to be noted that in vivo their relative local concentration and surface bound forms could influence the surface and receptor binding capacity of FH and FHRs, and thus cellular responses. Several questions remain to be answered regarding the non-canonical role of FH family members. The complement receptor CR3 (CD11b/CD18) and to a lesser extent CR4 (CD11c/CD18) were identified as the receptors for FH binding on immune cells; however, other candidates are also found, and the receptors for FHR proteins have not been clearly identified yet (14–16, 27, 58, 66). The similarity of FHR-1 domains 3-5 to FH domains 18-20 implies that the C-terminal SCR18-20 (or 19-20) domains are responsible for the binding and functional effect of FH and FHR-1 on immune cells. The identity of FHL-1 with FH SCR1-7 suggests, however, that SCR6-7 might play an important role as well in influencing immune cell functions. FHR-5 shows a lower degree of homology to both parts of FH, that may be the reason why we could not detect any direct functional effect of this molecule despite its prominent binding to the cells. These findings also suggest that FHR proteins could bind to other receptors than does FH.

While CR3 was identified as a FH receptor in the absence of deposited C3b (58) on human neutrophils and also on U937 monocytic cells ( Figure 3 , Supplementary Figure 1 ), the interaction of FH with cell surfaces is a complex phenomenon and is influenced among others by cell surface glycosaminoglycans and deposited other ligands, such as complement C3 fragments. The C-terminal domains of FH bind surface bound C3b via SCR19, while SCR20 binds sialic acids and glycosaminoglycans present on self surfaces which is important in the regulation of complement activation on the cell surface by FH (67–69); in addition, receptor-bound FH also retains its cofactor activity for C3b inactivation (16). The same C-terminal domains are involved in binding to neutrophils via complement receptors and mediating the non-canonical functions of FH (15). FH and FH SCR19-20 were also shown to prevent the adhesion of sickle cell disease erythrocytes to P-selectin and/or to CR3 by blocking C3b-cell interactions (70). Potentially, receptor-bound C3-fragments could also modify the interaction of FH and FHRs with cells. Moreover, it is unknown where exactly FH binds to its receptors. CR3 and CR4 are known to interact with several ligands, and their major ligand that promotes phagocytosis is iC3b on opsonized microbes and other particles (71). However, in our fibronectin and β-glucan induced NET model, iC3b used in combination with FH could not inhibit the effect of FH (16). Because there is evidence for ligand contact outside the I domain of CD18 (71) and the incubation of neutrophils with FH did not activate CR3 (72), it is likely that FH and iC3b bind differently. Further studies are warranted to better understand these interactions and their in vivo relevance.

In summary, we identify variable and context-dependent effects of FH family proteins on the function of human monocytes and neutrophil granulocytes. Furthermore, we show that FH-based complement inhibitors such as mini-FH, in addition to their effect on complement activation, may potentially also affect inflammatory cells. Further studies are needed to identify the exact receptors and mechanisms by which these proteins exert their functional effects on immune cells. A detailed characterization of the non-canonical functions of FH family proteins could provide a better understanding of their role in the clearance of different pathogens or host-derived debris and in the modulation of the resolution of inflammation and the pathogenesis of inflammatory disorders.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by National Research Ethics Committee (TUKEB ETT, permission number 838/PI/12). The patients/participants provided their written informed consent to participate in this study.

ÉK, MK, NS and MJ designed the experiments. ÉK, MK, NS, DH and AS performed experiments. MJ supervised the study. All authors discussed the data, revised and approved the manuscript. ÉK, MK, NS, AS and MJ wrote the manuscript. All authors contributed to the article and approved the submitted version.

This work was financially supported in part by the Hungarian National Research, Development and Innovation Office (OTKA grants K 109055 and K 125219, and VEKOP-2.3.3-15-2017-00021), the Hungarian Academy of Sciences (grants nr. LP2012-43 and 0106307), the Institutional Excellence Program to ELTE (NKFIH-1157/8/2019, D11206), the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 899163 (SciFiMed), and by the Kidneeds Foundation (Iowa, US).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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