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. Author manuscript; available in PMC: 2025 Apr 1.
Published in final edited form as: J Thromb Haemost. 2023 Dec 29;22(4):1215–1222. doi: 10.1016/j.jtha.2023.12.021

Interferons prime endothelium for toll-like receptor-mediated thrombin generation

Kelsey Sack 1,*, Nathan Eaton 1,*, Maneli Doroudian Tehrani 1, Robert Flaumenhaft 1
PMCID: PMC10960681  NIHMSID: NIHMS1955351  PMID: 38159649

Abstract

Background:

Respiratory infection is associated with microvascular thrombus formation and marked elevation of cytokines. The role of cytokines elaborated by pulmonary epithelium in thrombotic responses is poorly understood.

Aims:

Our goal was to identify cytokines of pulmonary epithelial cell origin that enhance thrombin generation in endothelium at concentrations equal to or less than those found in the circulation during infection.

Methods:

We screened multiple cytokines produced by pulmonary epithelium for the ability to enhance toll-like receptor (TLR)-mediated endothelial thrombin generation. Effects of cytokines on tissue factor and thrombomodulin expression, cytokine selectivity for different TLRs, and prothrombotic activity of endogenous cytokines in conditioned medium from pulmonary human epithelial cells were evaluated.

Results:

MIP-1β, MCP-1, IL-10, IL-6, IL-1β, TNFα, IFNα, IFNβ, and IFNγ were tested for their ability to enhance TLR3-mediated thrombin generation on endothelial cells. Only IFNs and TNFα promoted TLR3-mediated thrombin generation at levels that circulate during infection. IFNs robustly enhanced tissue factor expression when used in conjunction with TLR agonists and reduced thrombomodulin expression in endothelium independently of TLRs. IFNα, which is typically elevated with viral infection, only synergized with TLR3 agonists mimicking viral PAMPs. In contrast, IFNγ, which is typically in bacterial infection, synergized more effectively with TLR4 agonists released by bacteria. Conditioned media from inflamed pulmonary epithelial cells primed endothelium for TLR-mediated thrombin generation. Anti-IFN type I antibodies blocked this effect, indicating that endogenous IFNs prime endothelium for TLR-mediated thrombin generation.

Conclusion:

IFNs elaborated by pulmonary epithelium are necessary and sufficient to enhance TLR-mediated thrombin generation.

Keywords: interferons, toll-like receptors, tissue factor, endothelium, cytokine release syndrome

Introduction

Cytokine release syndrome is a well-recognized complication of severe inflammation and is associated with thrombosis. These complications are particularly prominent in respiratory infection. In this setting, the alveolus is first exposed to invading pathogens [1]. In its initial response, the pulmonary epithelium and pulmonary macrophages release an array of cytokines into the alveolar space and pulmonary circulation [1, 2]. With progressive invasion, the fragile barrier between alveolus and pulmonary capillaries breaks down. Sources of tissue factor (TF) in the circulation include monocytes and extracellular vesicles [3]. In addition, endothelial cells are exposed to pathogen-associated molecular patterns (PAMPs), which stimulate endothelial toll-like receptors (TLRs) [4] and transform the endothelium from a quiescent, antithrombotic surface to a more prothrombotic phenotype with decreased expression of thrombomodulin, glycocalyx shedding, exocytosis of Weibel-Palade bodies, and fibrin formation [5, 6]. However, the mechanisms linking vascular exposure to cytokines and PAMP with the prothrombotic state of endothelium are largely unknown. We therefore evaluated the effect of sequential exposure of endothelium to cytokines followed by PAMPs to model the effect of cytokines on endothelial phenotypes.

Methods

Human umbilical vein endothelial cells (HUVECs) or human lung microvascular endothelial cells (HMVEC-Ls) (Lonza, C2519A and CC-2527 respectively) monolayers (passage 3–7) were grown in EGM-2 complete growth media (Lonza, CC-3156 and CC-4176) to confluence in gelatin-coated (Stemcell Technologies, 7903) 96-well black plates (Greiner Bio-one, 655090). Human primary alveolar epithelial cells (HPAECs, Cellbiologics, H-6053) were seeded in gelatin-coated 6 well plates in the presence of complete SAGM media (Lonza, CC-3119 and CC-4124).

Coagulation assays

For cytokine panel assays, confluent HUVECs or HMVECs (93–94% blood vessel origin; 5–6% lymphatic origin) were incubated with listed cytokines (Biolegend: MIP-1β/554702, MCP-1/571402, IL-6/9570802, IL-1β/579402, TNFα/570102, IFNα/592704), IFNγ/570206; R&D Systems: IL-10/1064-ILB-010, IFNβ/8499-IF-010/CF, IFNε/9667-ME-025/CF, IFNω/11395–1) for 24 hours prior to 5 hour induction with poly(I:C) (Invivogen, tlrl-pic-5), poly(A:U) (Invivogen, tlrl-pau), CRX-527 (Invivogen, tlrl-crx527), or LPS (Sigma-Aldrich, L2630). For experiments involving endogenous alveolar cytokines, HPAECs were seeded at a density of 300K cells/well 6-well plates, incubated for 5 hours in the presence or absence of poly(I:C) (12.5 μg/ml), washed 3x with endotoxin-free PBS (Millipore sigma, TMS-012-A), and allowed to rest 24 hours in media. Conditioned media was subsequently incubated in the presence or absence of anti-type I IFN antibodies (including antibodies to IFNα, IFNβ, IFNω, IFNκ, IFNε; PBL Assay Science, 39000–1) or Type II IFN antibody (anti-IFNγ, PBL Assay Science, 31500–1) for 3 hours, and incubated on ECs for an additional 24 hours. ECs were then exposed to poly(I:C) (5 μg/ml) for 5 hours prior to assay. For thrombin generation experiments, cells were incubated with an 80–20% mixture of pooled human plasma and HBS, respectively, containing fibrin polymerization inhibitor H-Gly-Pro-Arg-Pro-OH (GPRP, 5 mM, Fisher, 50–382-505), 20 μl HEPES-buffered saline, pH 7.4 (HBS) and 0.7 to 5 mM CaCl2. Thrombin levels were measured using the SN-20 fluorogenic substrate (Haematologic Technologies, SN-20). Fluorescence was measured every minute for 60 minutes using a Synergy 4 plate reader (BioTek). For factor Xa (FXa) assays, cells were incubated with FX (125 nM Prolytix HCX-0050), FVIIa (0.6 nM Prolytix HCVIIA-0031) and FX chromogenic substrate (CS-11, 150 uM Aniara A229011) in HBS-BSA buffer supplemented with Ca2+. A SpectraMax spectrophotometer was utilized to monitor absorbance at 405 nM every minute for 60 minutes. Thrombin and FXa levels were determined by converting reaction rate to units/ml and nM/min, respectfully [7]. ECs were incubated with anti-TF antibody (Biomedica Diagnostics, 4509) for 10 minutes, prior to addition of buffer containing substrates.

qPCR Analysis

Confluent HUVECS were incubated with cytokines followed by poly(I:C) and a 2-step Cell-to Ct Taqman kit (Thermo Fisher Scientific 4399002) was utilized to measure transcript profiles using ThermoFisher Taqman gene expression probes: TM Hs00264920_s1, TLR3 Hs01551078_m1, TLR4 Hs00152939_m1, and 18s Hs99999901_s1. Gene expression was calculated via the ΔΔCT method.

Immunoblot analysis

Immunoblot analysis was performed as previously described [7]. Blots were blocked 60 minutes RT in 5% BSA with 0.1% tween-20 then probed with sheep anti-human TF antibody (Haematologic Technologies, PAHTF-S) and rabbit anti-human thrombomodulin (Abcam, ab109189) for 1 hours RT, and detected by secondary horseradish peroxidase-conjugated antibody.

Results and Discussion

To identify cytokines that contribute to the prothrombotic transformation of endothelium, we screened an array of alveolar epithelial cell-associated cytokines for their ability to influence thrombin generation on HUVECs in response to poly(I:C)-mediated TLR3 activation. MIP-1β, MCP-1, IL-10, IL-6, IL-1β, TNFα, IFNγ, IFNβ, and IFNα were evaluated. Incubation with several of the cytokines enhanced thrombin generation stimulated by poly(I:C). However, there was a clear distinction between IFNs and TNFα and the other cytokines. Dose curves showed that IFNs and TNFα were ~1000-fold more potent than the other cytokines at priming HUVECs for poly(I:C)-induced thrombin generation (Fig. 1A). In contrast, no cytokine except perhaps TNFα elicited detectable thrombin generation under the conditions of this assay (Fig. 1B). Although the prothrombotic effects of TNFα on HUVEC have been previously described [8], the prothrombotic effects of IFNs are not well-studied. IFN exposure followed by poly(I:C) markedly enhanced thrombin generation on HMVEC-L as well as on HUVECs, indicating that this effect was not selective to a single endothelial type (Fig. 1C). In addition to stimulating very high levels of endothelial thrombin generation, IFNs were able to stimulate thrombin generation at concentrations as low as 1–100 pg/ml, which are physiologically relevant in pulmonary infection [9]. Given the marked effect of IFN exposure on thrombin generation in endothelium, we evaluated mechanisms of this potent prothrombotic activity. An anti-TF antibody completely attenuated thrombin generation induced by either IFNα or IFNγ exposure followed by poly(I:C) stimulation (Fig. 1D). Consistent with a role for TF, exposure of HUVEC or HMVEC-L to IFNα or IFNγ alone did not stimulate factor VIIa-dependent FXa generation, but enhanced poly(I:C)-induced FXa generation (Fig. 1E).

Figure 1. IFNs prime endothelium for TLR3-induced thrombin generation.

Figure 1.

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(A) HUVECs were incubated with the indicated concentration ranges of the following cytokines: MIP-1β (16.7–500 ng/ml), MCP-1 (16.7–500 ng/ml), IL-10 (16.7–500 ng/ml), IL-6 (16.7–500 ng/ml), IL-1β (0.8–100 ng/ml), TNFα (12.5–800 pg/ml), IFNα (10–3000 pg/ml), IFNβ (1–30 pg/ml), and IFNγ (10–3000 pg/ml). Samples were incubated with the indicated cytokine for 24 hours and subsequently exposed to 10 μg/ml poly(I:C) for 5 hours. Thrombin generation on HUVEC monolayers was then tested. The dashed line indicates level of thrombin generation with PIC alone. Data indicate the mean±SEM of 6–12 samples. (B) HUVECs were incubated with buffer alone (NA), poly(I:C) (PIC, 10 μg/ml), MIP-1β (500 ng/ml), MCP-1 (500 ng/ml ), IL-10 (500 ng/ml ), IL-6 (500 ng/ml ), IL-1β (100 ng/ml), TNFα (200 pg/ml), IFNα (3 ng/ml), IFNβ (1 pg/ml), and IFNγ (3 ng/ml) for 24 hours. Thrombin generation on HUVEC monolayers was then tested. Data indicate the mean±SEM of 8–27 samples. (C) HMVEC-Ls were incubated with buffer alone (Ctrl), 100 ng/ml IFNα (IFNα), 100 ng/ml IFNβ (IFNβ), 100 ng/ml IFNγ (IFNγ) for 24 hours media or 24 hours of the indicated additions, followed by a 5-hour incubation with media or 10 μg/ml poly(I:C) (PIC). Thrombin generation on HMVEC-L monolayers was then tested. Data indicate the mean±SEM of 6 samples. (D) HUVECs were incubated with media alone, 100 ng/ml IFNα, or 100 ng/ml IFNγ. After 24 hours, samples were exposed to vehicle (Control, IFNα, IFNγ) or 10 μg/ml poly(I:C) (PIC; IFNα, then PIC; IFNγ, then PIC). At the termination of the experiment samples were exposed to non-immune IgG (NI IgG) or anti-TF antibody (anti-TF ab). Samples were then evaluated for thrombin activity. Data represent the mean±SEM of 9 samples.(E) HUVECs and HMVEC-Ls were incubated with media alone, 100 ng/ml IFNα, or 100 ng/ml IFNγ. After 24 hours of media or IFN treatment, samples were exposed to vehicle (Control, IFNα, IFNγ) or 10 μg/ml poly(I:C) (PIC; IFNα, then PIC; IFNγ, then PIC). Samples were evaluated for FXa generation. ****p < 0.001 (1-way ANOVA, PIC compared with IFNα+PIC or IFNγ+PIC).

Studies to evaluate the mechanism of IFN-induced priming showed that although exposure of HUVECs to IFNs did not itself enhance TF expression, IFNs significantly augmented poly(I:C)-mediated TF expression (Fig. 2A,B). IFNα and IFNγ significantly inhibited thrombomodulin expression, even in the absence of poly(I:C) (Fig. 2A,C). Evaluation of HMVEC-L showed similar results (Fig. 2D-F). PCR of HUVEC samples showed a decrease in thrombomodulin transcripts following exposure to IFNγ compared with IFNα (Fig. 2G). Poly(I:C) exposure also inhibited thrombomodulin expression [6]. Together, these data indicate that IFNs promote a pro-thrombotic endothelium by augmenting poly(I:C)-mediated TF upregulation and by thrombomodulin downregulation involving decreased production and additional undefined mechanisms.

Figure 2. IFNs promote TLR-induced TF expression and reduction of thrombomodulin expression.

Figure 2.

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(A) HUVECs were incubated with no addition, IFNα, or IFNγ for 24 hours and then exposed to either buffer (Ctrl, IFNα, IFNγ) or poly(I:C) for 5 hours (PIC; IFNα, PIC; IFNγ, PIC). Tissue factor (TF) and thrombomodulin (TM) levels were then analyzed by Western blot analysis and quantified using densitometry (B, C). Error bars represent SEM of 3 samples. * p < 0.05, ** p < 0.01, **** p < 0.001 (1-way ANOVA). (D) HMVEC-Ls were incubated as described in (A). Tissue factor (TF) and thrombomodulin (TM) levels were then analyzed by Western blot analysis and quantified using densitometry (E, F). Data represents mean +/− SEM of 3–5 samples. *p<0.05, ** p < 0.01, **** p < 0.001 (1-way ANOVA). (G) HUVECs were incubated with no addition, IFNα, or IFNγ for 24 hours and then exposed to either buffer (Ctrl, IFNα, IFNγ) or poly(I:C) for 5 hours (PIC; IFNα, PIC; IFNγ, PIC) prior to cell lysis for qPCR of thrombomodulin gene expression levels. The median of control samples was utilized as the reference value. Data is represented as mean +/− SEM of 9 samples. *** p < 0.01, **** p < 0.001 (1-way ANOVA).

Endothelial cells express many TLRs that could potentially elicit prothrombotic changes. To evaluate whether or not IFNs enhance thrombin generation in endothelium stimulated through TLRs other than TLR3, we exposed endothelium to IFNα and IFNγ, followed by stimulation with different TLR-selective agonists. IFNγ exposure enhanced thrombin generation on endothelium in response to the TLR1/2 agonist PAM3CSK4, but IFNα did not have a significant effect (Fig. 3A). In contrast, the stimulation of endothelial thrombin generation by the TLR3 agonists, poly(I:C) and poly(A:U), was more potently enhanced by IFNα than IFNγ (Fig. 3B). Stimulation of thrombin generation by TLR4 agonists, including LPS and CRX-527, was significantly enhanced by IFNγ, but not IFNα (Fig. 3C). To evaluate the mechanism by which IFNs enhance responsiveness to TLRs, we monitored TLR3 and TLR4 transcripts following exposure to either IFNα or IFNγ. These studies showed that IFNα exposure preferentially increased TLR3 but not TLR4 transcripts, while IFNγ exposure increased both (Fig. 3D).

Figure 3. IFNs prime thrombin generation induced by different TLRs.

Figure 3.

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(A-C) HUVECs were incubated with either buffer alone (Control), IFNα (IFNα), or IFNγ (IFNγ) for 24 hours. ECs were then stimulated with 1 μg/ml of either (A) the TLR1/2 agonist PAM3CSK4, (B) TLR3 agonists, poly(I:C) (PIC) or poly(A:U) (PAU), or (C) TLR4 agonists, LPS or CRX-527, as indicated. Samples were subsequently evaluated for thrombin generation. Data indicate the mean±SEM of 9–43 samples. * p < 0.05, ** p < 0.01, **** p < 0.001 (1-way ANOVA). (D) HUVECs were incubated with no addition, IFNα, or IFNγ for 24 hours and then exposed to buffer for 5 hours prior to cell lysis and qPCR of TLR3 and TLR4 gene expression levels. The median of control samples was utilized as the reference value. Data indicate the mean +/− SEM of 9 samples. ***p< 0.001, **** p< 0.0001 (2-way ANOVA).

Type I IFNs include 16 distinct subtypes that all bind to the type I IFN receptor [10]. Type I IFNs produced by inflamed alveoli include IFNα, IFNβ, IFNε, and IFNω. We evaluated the bias of these Type I IFNs with regard to priming through TLR3- versus TLR4-mediated pathways. All type I IFNs augmented TF activity stimulated through TLR3 via poly(IC) without significantly priming TF activity stimulated through TLR4 via LPS (Fig. 4A). To evaluate whether pulmonary epithelial cells are able to secrete endogenous IFNs that can directly promote prothrombotic EC activity, we tested conditioned media from pulmonary epithelial cells. These studies showed that media from inflamed pulmonary epithelial cells had only a small, non-statistically significant effect on endothelial thrombin generation by itself, but primed endothelium for substantial thrombin generation in response to poly(I:C) (Inflamed, PIC; Fig. 4B). Media conditioned from quiescent epithelial showed only a trend towards priming endothelial thrombin generation compared to unconditioned media (Quiescent, PIC; Fig. 4B). To determine whether or not type I and type II IFNs are responsible for the procoagulant priming activity produced by inflamed epithelial cells, we evaluated the effect of IFN neutralizing antibodies on the priming activity in inflamed conditioned media. Anti-type I IFN antibodies (a mixture of antibodies that neutralize type I IFNα, β, ε, and ω) substantially decreased the priming effect of inflamed condition media, whereas anti-type II IFN antibody showed less substantial inhibition of priming by inflamed condition media. The combination of anti-type I IFN plus anti-type II IFN antibodies decreased the priming effect of inflamed condition media in a manner similar to anti-type I IFN antibodies alone (Fig. 4B).

Figure 4. Endogenous IFNs prime endothelial cells for TLR3-induced thrombin generation.

Figure 4.

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(A) HUVECs were incubated with either media alone (Control) or 100 ng/ml IFNα (IFNα), IFNγ (IFNγ), IFNε (IFNε), or IFNω (IFNω) for 24 hours. ECs were then stimulated vehicle (Con), poly(I:C), or LPS in the presence of LBP and CD14. Subsequently, samples were evaluated for FXa generation. Error bars represent mean +/− SEM for 9 samples each. **** p < 0.001 (1-way ANOVA) (B) HPAECs were grown to confluence and exposed to either buffer alone (i.e., for generating quiescent conditioned media) or 12.5 μg/ml poly(I:C) (i.e, for generating inflamed conditioned media) for 5 hours. Cells were then washed 3x to remove poly(I:C) and incubated with fresh media for 24 hours, after which conditioned media were collected. HUVECs were subsequently incubated with either unconditioned media alone (Fresh), quiescent conditioned media (Quiescent), or inflamed conditioned media (Inflamed). For samples including antibodies, inflamed condition media was first exposed to either non-immune IgG (IgG), anti-type I IFN antibodies (α-type I IFN), anti-type II IFN antibodies (α-type II IFN), or both antibody types together (α-type I IFN, α-type II IFN), as indicated, and transferred to HUVECs. Following a 24-hour incubation with different medias, HUVECs were exposed to either buffer (no PIC) or 5 μg/ml poly(I:C) for 5 hours (PIC), as indicated. HUVECs were then evaluated for thrombin generation. Data indicate the mean±SEM of 10–12 samples. * p < 0.05, ** p < 0.01, **** p < 0.001 (1-way ANOVA). (C) Initial infection (priming) - The initial response to pulmonary infection is the elaboration of cytokines, including IFNα and IFNγ, by alveolar macrophages and pulmonary epithelial cells [2]. IFNα production is stimulated by viral infections and primarily increases expression of TLR3 (blue) in endothelium [27]. IFNγ production is stimulated by bacterial infections and increases endothelial expression of TLR3 (blue) and TLR4 (red) [28]. Both IFNs inhibit thrombomodulin expression (purple). Progressive infection - With progression of the infection, the barrier separating pathogens from the circulation breaks down and endothelium is exposed to PAMPs. Viral PAMPs stimulate TLR3 (blue) and bacterial PAMPs stimulate TLR4 (red). Stimulation of either TLR type results in upregulation of TF, FXa generation, and cleavage of prothrombin into thrombin (purple). (Components of the figure were created using Biorender).

Cytokine release syndrome, or ‘cytokine storm’, occurs in the setting of both bacterial and viral infections [11]. Although cytokine storm has been associated with clinical thrombosis [12], the mechanistic underpinnings of this association and the primary cytokines involved remain poorly understood within the context of aberrant coagulation. Here, we screened a range of common cytokines produced by pulmonary epithelium and have identified a role for epithelial alveolar-derived IFNs in promoting procoagulant activity in endothelium. Specifically, IFNs are three-orders of magnitude more potent than any other cytokine tested except TNFα at priming endothelium for TLR-mediated thrombin generation (Fig. 1A). The effect of IFNs on the endothelial prothrombotic transformation is notable for both the extent of thrombin generation that they elicit and the low concentration at which they function. Under the conditions of our assay, no other cytokines except TNFα were effective in physiologic, pg/ml doses (Fig. 1). Other pulmonary cytokines (notably TNFα and IL-1β) transiently enhance TF expression on ECs [8, 13–15] and these may also be prothrombotic. IL-6 has been implicated in the pathogenesis of thrombosis and endothelial cell dysfunction [16, 17]. However, in the context of our experimental set-up, the fact that antibodies to IFNs completely inhibit TLR-mediated thrombin generation (Fig. 4) together with the effect of purified IFNs on TLR-stimulated procoagulant activity demonstrate that this family of cytokines is both necessary and sufficient to prime endothelium for prothrombotic changes mediated by TLRs.

Animal studies and clinical experience support the premise that IFNs are prothrombotic in the setting of infection. Mice lacking the IFNα/β receptor 1 showed markedly reduced thrombus formation in lung and liver following challenge with LPS or cecal ligation and puncture [18]. Circulating markers such as thrombin-antithrombin, PAI-1, and D-dimer were also reduced in IFNα/β receptor 1-null mice [18, 19]. Thrombus size was decreased in IFNγ−/− mice subjected to IVC ligation [20]. Mice lacking TIR-domain-containing adaptor-inducing interferon-β (TRIF), which induces generation of type I IFNs, were also protected from bacterial-induced thrombosis [21, 22]. Clinical use of recombinant IFN is associated with increased prothrombotic markers in patients with myeloproliferative disease [23]. Furthermore, IFNα therapy is associated with thrombotic microangiopathy secondary to endothelial dysfunction [24, 25] and with retinal vein occlusion [26].

Our findings provide a new framework for understanding the role of IFNs in cytokine-related thrombus formation (Fig. 4C). In the initial stages of respiratory infection, IFNs are elevated and prime the endothelium by decreasing expression of thrombomodulin and increasing expression of TLRs (Fig. 2, 4C) [27, 28]. With progressive infection, the endothelium is increasingly exposed to PAMPs, resulting in enhanced TF expression with ensuing generation of FXa and thrombin (Fig. 4C). IFNα subtypes predominate in the setting of viral infections and synergize with TLR3 agonists, such as viral double-stranded RNA, to stimulate thrombin generation in endothelium. IFNγ, the elevation of which is more pronounced in bacterial and parasitic infections, synergizes with TLR2/4 agonists, such as LPS (Fig. 3, 4C) [29, 30]. Future studies will evaluate the signaling pathways that underlie the specificity of IFN priming mechanisms and will determine whether targeting IFN is useful in preventing DIC, microvascular thrombosis, and other thrombotic syndromes associated with infection.

Acknowledgment

This work was supported by the National Institutes of Health grants R35HL135775 (R.F.), U01HL143365 (R.F.), and T32HL007917 (N.E., K.S., R.F.).

Footnotes

Conflict of Interest Disclosure

R.F. is a founder and consultant for Platelet Diagnostics. The other authors declare no competing financial interests.

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