Cleavage of Fibrinogen by Proteinases Elicits Allergic Responses Through Toll-Like Receptor 4

Abstract

Proteinases and the innate immune receptor Toll-like receptor 4 (TLR4) are essential for expression of allergic inflammation and diseases such as asthma. A mechanism that links these inflammatory mediators is essential for explaining the fundamental basis of allergic disease but has been elusive. Here, we demonstrate that TLR4 is activated by airway proteinase activity to initiate both allergic airway disease and antifungal immunity. These outcomes were induced by proteinase cleavage of the clotting protein fibrinogen, yielding fibrinogen cleavage products that acted as TLR4 ligands on airway epithelial cells and macrophages. Thus, allergic airway inflammation represents an antifungal defensive strategy that is driven by fibrinogen cleavage and TLR4 activation. These findings clarify the molecular basis of allergic disease and suggest new therapeutic strategies.

Allergic asthma is a chronic inflammatory airway disease that is characterized by both airway obstruction and enhanced systemic and airway allergic inflammation marked by interleukin-4 (IL-4)–secreting T helper 2 (T_H2) cells, eosinophils, and serum immunoglobulin E (IgE). Proteinases able to elicit T_H2 cell–driven allergic responses are secreted by fungi (1) and can be found in natural sources linked to allergic disease such as pollens (2) and dust mite antigens like Der p 1 (3, 4). Nonetheless, nonproteinase allergens such as ovalbumin also possess allergenic activity. Prior studies have linked TLR4, a microbial pattern-recognition receptor, to both proteinase-dependent and -independent allergic responses in mice (5–7), but a mechanism that explains the importance of proteinases and TLR4 in diverse allergic contexts involving both proteinase-active and -inactive allergens remains unknown.

To study the role of TLR4 in the context of proteinase-dependent allergic inflammation, we assessed wild-type (WT) and Tlr4^−/− mice after intranasal exposure to a fungal proteinase derived from Aspergillus oryzae (PAO) [endotoxin content: 1.7 × 10⁻³ endotoxin units (EU)/μg]. Consistent with our prior observations (2), WT mice challenged with PAO developed canonical features of asthma, including airway hyperresponsiveness, airway infiltration by eosinophils, enhanced production of the mucin gene transcript Muc5ac, goblet cell metaplasia of the airway, and enhanced production of transcripts for the proinflammatory cytokines IL-4, IL-5, and IL-13 (fig. S1, A to G). In contrast, all of these allergic parameters, with the exception of lung IL-4 transcripts, were either attenuated or abrogated in Tlr4^−/− mice. Thus, TLR4 is essential for the expression of proteinase-dependent asthma-like disease in mice.

Quantification of IL-4–secreting cells from whole lungs of proteinase-challenged mice (fig. S1H) confirmed the equivalent presence of IL-4– producing cells in the lung regardless of mouse genotype, suggesting that T_H2 cell development and recruitment occurred independently of TLR4. We then immunized mice against ovalbumin and confirmed that ovalbumin-specific T_H2 cell development was equivalent or enhanced in Tlr4^−/− mice compared with controls (fig. S1I). Induction of total IgE, an IL-4– and T_H2 cell–dependent process (8–11), was also identical between WT and Tlr4^−/− mice (fig. S1J).

Both the fungus A. niger and proteinase-free ovalbumin (1.8 × 10⁻³ EU/μg) also induced TLR4-dependent allergic lung disease in controls compared with proteinase-challenged WT mice (fig. S2). The consistent defect in disease expression seen in Tlr4^−/− mice was durable because identical reductions in allergic disease parameters were seen after 2 and 4 weeks of ovalbumin immunization (fig. S2). Thus, TLR4 was required for the development of allergic airway disease, regardless of allergen proteinase content, but was dispensable for T_H2 responses.

We confirmed the reduced IL-5 transcript production in Tlr4^−/− mice by assessing secreted IL-5 levels in bronchoalveolar lavage fluid (fig. S3A). Type 2 innate lymphoid cells (ILCs) secrete IL-5 and IL-13, but not IL-4, in the setting of airway proteinase challenge (12), suggesting that these cells might be influenced by TLR4. Indeed, relative to vehicle-challenged animals, ILCs failed to be recruited as robustly into bronchoalveolar lavage fluid in Tlr4^−/− mice relative to WT animals after proteinase challenge, potentially accounting in part for the reduced T_H2 cytokine production in Tlr4^−/− mice (fig. S3, B and C).

We next considered whether other TLRs played a role in proteinase-dependent allergic lung disease. Most TLRs signal through one of two major adapter proteins, MyD88 and TRIF, whereas TLR4 signals through both adapters (13). Mice deficient in either MyD88 or TRIF showed an enhanced or identical disease phenotype as genotype matched control mice when challenged with A. niger spores (fig. S4). In contrast, mice deficient in both MyD88 and TRIF showed complete disease abrogation. Thus, proteinase-dependent allergic lung disease is mediated through TLR4 and not other TLRs.

Because TLR4 does not determine T_H2 responses, we turned to macrophages to further explore how TLR4 controls allergic disease. Relative to naïve cells, bone marrow–derived macrophages (BMDMs) expressed distinct transcriptional programs when activated by lipopolysaccharide (LPS), interferon- γ (IFN-γ), and PAO (Fig. 1A). Specific genes induced by PAO included lysozyme (Lyz), macrophage receptor with a collagenous structure (Marco), and secretory leukoproteinase inhibitor (Slpi), all of which have been linked to antifungal immunity (14–16), and their induction was dependent on TLR4 (Fig. 1B). PAO did not induce genes linked to previously characterized macrophage phenotypes, including IFN-γ–activated type 1 macrophages (M1) (nitric oxide synthase 2 (NOS2)] and IL-4–activated M2 (arginase 1 and FIZZ1) (17) (Fig. 1, C and D). Alveolar macrophages from mice treated with PAO similarly showed up-regulation of lysozyme, MARCO, and SLPI (Fig. 1E). Thus, PAO induced a macrophage phenotype marked by expression of a distinct transcriptional profile that included genes with antifungal properties.

Fig. 1. PAO induces a distinct fungistatic macrophage phenotype through TLR4.

(A to D) BMDMs from WT or Tlr4^−/− mice were left unstimulated (N) or cultured in the presence of lipopolysaccharide (L), IFN- γ (I), PAO (P), or IL-4 (4) as indicated. (A) Heat map depicting the relative expression of 252 gene probes, as assessed by microarray (P < 0.01 and fold change >1.5, comparing PAO to each of the other groups) of WT BMDMs. (B) Polymerase chain reaction (PCR)–based analysis of lysozyme, MARCO, and SLPI. (C) NOS2 expression in IFN- γ- and PAO-activated WT macrophages. (D) Arg1 and Fizz1 expression in IL-4– and PAO-activated WT macrophages. (E) MARCO, SLPI, and lysozyme (LYZ) mRNA expression in alveolar macrophages derived from mice challenged with PBS (S) or PAO (P). (F) BMDMs from WT (top row) and Tlr4^−/− (bottom row) mice were treated with IFN- γ, LPS, IL-4, or PAO for 24 hours and then cultured with A. niger conidia. Photomicrographs depict filamentous fungal growth. (G) Quantification of fungal growth in the same experiment, as assessed by XTT assay (n = 3 replicates per group). (H) PAO-treated human peripheral blood monocyte-derived macrophages were cultured under the same conditions as in (F) and assessed by XTT assay for their ability to restrain fungal growth (n = 3 patients). (I) WT and Tlr4^−/− mice were intranasally challenged with 400,000 A. niger conidia, and lungs were harvested and fungal colony forming units (CFU) were determined over 7 days. (J) Fungistatic potential of BMDMs was determined as in (G), but in the presence (+) and absence (−) of fetal bovine serum (FBS). Data are presented as means ± SEM (error bars) from one of three comparable experiments. *P < 0.05; **P < 0.01; ***P < 0.001 by Mann-Whitney (two group comparisons) and Kruskal-Wallis (three or more group comparisons) tests.

We next determined if PAO-activated macrophages were capable of restraining fungal growth in vitro. Relative to naïve BMDMs, as assessed by both microscopy and colorimetric quantification, only IFN-γ– and PAO-activated macrophages efficiently controlled fungal growth when the conidia of A. niger were added to cultures (Fig. 1, F and G, and fig. S5). Human monocyte-derived macrophages were similarly responsive to PAO treatment, although less so to IFN-γ compared with mouse BMDMs (Fig. 1H). However, control of fungal growth through PAO, but not IFN-γ, required the presence of TLR4 (Fig. 1, F to H). Again, MyD88 and TRIF were individually dispensable for control of fungal growth in macrophages activated by PAO, but deletion of both adapters abrogated the ability of mouse macrophages to control fungal growth (fig. S6). These in vitro findings correlated with a reduced ability of Tlr4^−/− mice to clear A. niger conidia from the airway after a single inhalational challenge (Fig. 1I).

Unlike other macrophage activators, PAO-dependent inhibition of fungal growth (fungistasis) required the presence of serum, suggesting that fungal proteinases acted through both a serum factor and TLR4 to induce macrophage antifungal immunity (Fig. 1J). Fibrinogen, a proposed TLR4 ligand (18), is the functional mammalian analog of the arthropod factors pro-Späetzle and coagulogen, which regulate anti-fungal immunity through Toll (19).

To determine if fibrinogen mediates proteinase-dependent fungistasis, we added it (1.8 × 10⁻⁵ EU/μg) to BMDM cultures, with and without fungal and endogenous proteinases, and the conidia of A. niger. Only when stimulated by PAO in the presence of serum did BMDMs exhibit robust fungistatic activity (Fig. 2A). Identical results were obtained in experiments in which PAO was substituted with the endogenous proteinase thrombin, which converts fibrinogen to fibrin as the terminal step in the clotting cascade while also creating additional cleavage products that do not participate in clot formation (Fig. 2B). These results suggested that rather than fibrinogen in per se, fibrinogen cleavage products (FCPs) were required to induce fungistasis. FCPs created by incubating fibrinogen with PAO or thrombin induced fungistasis to a comparable degree as whole serum and proteinase when added to BMDMs (Fig. 2, A and B). Moreover, the thrombin inhibitor hirudin (3.4 × 10⁻³ EU/μg) neutralized both PAO-and thrombin-dependent fungistasis that was induced in the presence of serum (Fig. 2C). Another abundant serum protein and putative TLR4 ligand, fibronectin (20), had no effect on macrophage fungistasis in either native or cleaved forms (fig. S7).

Fig. 2. Fibrinogen mediates PAO-dependent fungistasis through TLR4.

BMDMs were cultured for 24 hours in the presence of FBS, PAO, fibrinogen (FG), FCP, or thrombin (THR), as indicated, and then inoculated with the conidia of A. niger for 24 hours, testing the requirement of (A) PAO and (B) thrombin for induction of fungistasis and (C) the effect of the thrombin inhibitor hirudin on PAO- and thrombin-dependent fungistasis, as assessed by XTT assay. (D) PAO and (E) thrombin were further compared with FCPs alone for their ability to induce fungistasis in the presence or absence of the Tlr4 gene (n = 3 replicates per group). Data are presented as means ± SEM (error bars) from one of four comparable experiments. *P < 0.05 by Mann-Whitney test.

In addition to fungistasis (Fig. 2, D and E), FCPs also induced in BMDM the expression of mRNA for IL-13Rα1, a component of the IL-13 receptor that is required for expression of allergic airway disease (21), and the airway mucin gene Muc5ac through TLR4 (Fig. 3A). FCPs yielded similar findings and also induced fungistatic activity in human primary airway epithelial cells (Fig. 3, B and C).

Fig. 3. FCPs up-regulate IL-13ra1 and Muc5AC on airway epithelium.

(A) WT and Tlr4^−/− mouse primary airway epithelial cells were cultured in the presence of IL-13 or FCPs for 24 hours, and Il13ra1 and Muc5ac gene transcripts were analyzed by quantitative PCR (n = 3). (B)Il13ra1 and Muc5ac transcripts were similarly analyzed from human primary airway epithelial cells 24 hours after treatment with FCPs or left unstimulated (Un) (n = 4). (C) Unstimulated and FCP-pretreated human airway epithelial cells were assessed for their ability to inhibit the growth of A. niger by XTT assay (n = 3 replicates per group.). Data are presented as means ± SEM (error bars) from one of three (murine) or two (human) comparable experiments. *P < 0.05; ***P < 0.001 by Mann-Whitney test.

Together, these findings support a model in which both endogenous and exogenous airway proteinase activities with allergenic potential produce alternate TLR4 ligands from fibrinogen that license innate immune cells to respond to T_H2 cells, as required for full expression of allergic airway disease. To test this model, we first administered intranasally to mice the maximum tolerated dose of FCPs, 0.6 mg per dose (Fig. 4A), which induced modest airway eosinophil recruitment and Muc5ac gene expression but failed to induce airway hyperresponsiveness and IL-4– secreting cells (Fig. 4, B to F). Thus, FCPs appeared to influence only innate immune cells and specifically did not induce T_H2 responses that are required for robust allergic lung disease.

Fig. 4. Fibrinogenolysis is necessary but insufficient for expression of robust allergic airway disease.

(A) C57BL/6 mice were challenged intranasally with PAO or FCPs as indicated, after which (B) airway hyperresponsiveness, (C) total bronchoalveolar lavage fluid (BALF) inflammatory cells, (D) lung Muc5AC transcripts, and (E) total lung IL-4– secreting cells were quantitated. (F to J) C57BL/6 mice were intranasally challenged with PAO without and with hirudin or hirudin alone on alternating days for 2 weeks, and the indicated parameters were assessed (n ≥ 3 mice per group). Data are presented as means ± SEM (error bars) from one of three comparable experiments. Data are averages ± SEM. *P < 0.05; ***P < 0.001 by Kruskal-Wallis test. R_RS, respiratory system resistance.

We conducted additional studies to confirm that airway proteinase activity was required for allergic lung disease using the proteinase inhibitor hirudin. Our in vitro studies indicated that hirudin, a known thrombin antagonist, could also inhibit PAO-mediated fungistasis (Fig. 2C), suggesting that hirudin may possess broad-spectrum antiproteinase activity. In a dose-dependent manner, hirudin progressively and significantly attenuated PAO-dependent allergic lung disease while leaving unaffected robust lung IL-4 responses (Fig. 4, G to K), a phenotype that resembles that of Trl4^−/− mice challenged with diverse allergens (figs. S1 and S2). Hirudin further inhibited ovalbumin-dependent allergic airway disease, suggesting that ovalbumin challenge activates an endogenous proteinase, possibly thrombin, to achieve the fibrinogenolysis that is necessary for disease expression (fig. S8). Together, these studies confirm the importance of airway fibrinogenolysis for the expression of allergic lung disease, regardless of the proteinase content of the inhaled allergen.

Although previous studies have shown that TLR4 contributes to T_H2 responses (5, 22, 23), we have shown here that TLR4 is not required for T_H2 cell development but rather is required for responsiveness of innate airway cells to T_H2 cells. Our findings do not exclude the possibility that bacterial endotoxin, a canonical TLR4 ligand, could mediate T_H2 responses under some conditions, as shown previously (5), but additional studies are needed to determine the contribution of fibrinogenolysis to this observation. Although independent of TLR4, T_H2 cells nonetheless develop through a proteinase-dependent pathway (24), suggesting that proteinases coordinate both innate and adaptive allergic pathways that together lead to allergic inflammation and disease (fig. S9).

Ultimately, mammalian TLR4 preserves the crucial role of arthropod Toll by linking proteinase-dependent fibrinogenolysis to antifungal immunity. However, in addition to fungi, mammals must also defend against other proteinase-associated pathogens such as helminth parasites (25) and, potentially, viruses (26). Although highly effective, the ancient proteinase-Toll–based defensive strategy is also susceptible to aberrant activation in response to innocuous proteinase sources such as pollens and many allergens, both with and without intrinsic proteinase activity. Clarification of the contribution of true infections to common allergic airway disorders such as allergic rhinitis, asthma, and chronic rhinosinusitis will determine the usefulness of interrupting FCP-TLR4 signaling as a therapeutic strategy.

Supplementary Material

Figure S1. TLR4 is required for allergic airway disease but not Th2 responses. Wildtype C57BL/6 and TLR4 knockout (Tlr4−/−) mice were intranasally challenged with PAO dervied from A.oryzae or PBS daily over two weeks after which (A) airway responsiveness to acetylcholine provocation, (B) total airway inflammatory cells (eosinophils:EOS; macrophages: MACS; neutrophils: NEUTS; lymphocytes: LYMPH), and (C) lung Muc5AC gene expression were quantified. (D) Representative lung histology of the same mice. (E-G) Quantitative real-time PCR quantitation of transcripts for IL-4, IL-5, and IL-13 from whole lung, respectively. (H) Total IL-4-secreting cells from whole lung. WT and Tlr4−/− mice were immunized with ovalbumin (OVA) weekly for 3 weeks after which (I) OVA-specific IL-4-secreting splenocytes were quantitated by ELISpot and (J) total serum IgE responses were determined. Data are means ± S.E.M. from one of three comparable experiments*, P<0.05; **, P<0.01; ***, P< 0.001 by Kruskal-Wallis test (n=5/group). Arrows point to mucus-producing goblet cells.

Figure S2. TLR4 is required for allergic airway disease due to diverse allergens. (A-F) Airway hyperesponsiveness (A,D,G), bronchoalveolarlavage fluid (BALF) inflammatory cells (B,E,H; eosinophils:EOS; macrophages: MAC; neutrophils: NEUT; lymphocytes: LYMPH) and lung IL-4-secreting cells (C,F, I) after immunization with ovalbumin (OVA) for two weeks (A-C) or four weeks (D-F) and intranasal challenge with OVA or PBS for four consecutive days, comparing responses of wild type (+/+) and TLR4−/− mice. (G-I) Wild type and TLR4−/− mice were challenged intranasally with the conidia of Aspergillus niger (AN) (AN) daily for 16 days after which the same parameters as in A-C were determined. Data are presented as means ± S.E.M. and are representative of at least 3 independent experiments for each allergen. ***: P<0.001 by Kruskal-Wallis test, (n=5/group).

Figure S3. Reduced ILC recruitment into bronchoalveolar lavage fluid after proteinase challenge. Wild type (WT) and TLR4-deficient mice were challenged with PAO intranasally daily for 14 days after which bronchoalveolar lavage fluid was collected for quantitating IL-5 (A) and innate lymphoid cells (ILC; B,C). B. Gating strategy for ILC. C. Aggregate data comparing the fold change in BALF ILC in wild type (WT) and Tlr4−/− mice. Data are presented as means ± SEM and are representative of two independent experiments. *: P < 0.05; ***: P < 0.001 by Mann-Whitney test, n=4 mice/group.

Figure S4. Requirement of MyD88 and TRIF for proteinase-mediated allergic lung disease. Mice deficient in MyD88 (A-D), TRIF (D-F) and both MyD88 and TRIF (DKO; G-I) were challenged daily with PAO for 14 days and then assessed respectively for airway hyperesponsiveness, bronchoalveolar lavage fluid (BALF) inflammatory cells (eosinophils:EOS; macrophages: MAC; neutrophils: NEUT; lymphocytes: LYMPH) and lung IL-4-secreting cells. Data are presented as averages ± S.E.M. and are representative of three independent experiments for each mouse genotype. *: P<0.05; **: P<0.01; ***: P<0.001 by Kruskal-Wallis test (n=5/group).

Figure S5. IFN-γ and PAO induce fungistasis in macrophages. Mouse BMDM were cultured for 24 hours as naive cells or in the presence of IFN-γ or PAO after which the the conidia of A. niger were added and the cells incubated for 24 more hours at distinct macrophage (Mac):conidia ratios. Fungistasis was then determined by XTT assay. Data are means ± S.E.M. and are representative of two independent experiments, *, P<0.01. (n=3/group), Kruskal-Wallis test.

Figure S6. MyD88 and TRIF are together required for proteinase-dependent fungistasis. BMDM were cultured for 24 hours under naive and proteinase activating conditions with PAO and then inoculated with the conidia of Aspergillus niger for 24 hours and fungal growth was assessed by XTT assay. Data are means ± S.E.M. and are representative of three independent experiments. *, P<0.01. (n=3/group), Mann-Whitney test. NS: not significant.

Figure S7. Fibronectin lacks fungistatic activity acting on macrophages. BMDM were cultured with and without fetal bovine serum (FBS), proteinase of Aspergillus oryzae (PAO) and fibronectin as indicated and the effect on fungal growth was determined by XTT assay. Data are means ± S.E.M. and are representative of two independent experiments. **: P<0.01, (n=3/group), Kruskal-Wallis test.

Figure S8. Hirudin blocks ovalbumin-depenent allegic lung disease. C57BL/6 mice were immunized with ovalbumin weekely for two weeks and then challenged intranasally with ovalbumin in the presence or absence of increasing amounts of hirudin on alternating days for 2 weeks. Mice were assessed for (A) airway hyperresponsiveness, (B) bronchoalveolar lavage fluid (BALF) inflammatory cells, (D) Muc5AC mRNA induction and (D) total lung IL-4-secreting cells. Data are means ± SEM and are respresentative of two independent experiments. * P<0.05; **, P<0.01; ***, P<0.001 by Kruskal-Wallis test (n=3 or 4 mice/group).

Figure S9. Proteinase model of allergic airway disease pathogenesis. The healthy lung routinely encounters low-grade proteinase activity through inhalation of fungi, pollens and other proteinase sources. Enhanced proteinase exposure, i.e., through airway surface mycotic infections (ASMI), causes lung damage that may include enhanced airway epithelial and vascular endothelial cell permeability that promote release of fibrinogen into the airway interstitium and lumen. Airway epithelial cells may also secrete fibrinogen directly into the airway (29). Exogenous proteinases, but also endogenous thrombin, promote fibrinogen cleavage and the generation of both fibrin clots and fibrinogen cleavage products (FCP) that function as alternate ligands for TLR4, triggering the activation of distinct innate immune cells, including airway epithelium and macrophages. Enhanced anti-fungal immunity induced in FCP-activated innate immune cells in the activated lung promotes resolution of ASMI, and simultaneously increased responsiveness to the cytokines of Th2 cells, e.g., IL-13, through increased expression of the IL-13 receptor subunit IL-13Rα1. Simultaneously, through a non-TLR4-dependent mechanism, airway proteinases initiate the differentiation of Th2 cells (24), which also promote anti-fungal immunity (30). Unchecked airway proteinase activity, i.e., through unresolved ASMI or overwhelming proteinase exposure, leads to allergic airway diseases such as asthma in which FCP-dependent innate immune and FCP-independent Th2 responses (31) contribute equally.