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Article Role of TLR4 Complex in the Regulation of the Innate by

Mingzhe Zheng, Anthony Ambesi and Paula J. McKeown-Longo * Department of Regenerative & Cell Biology, Albany Medical College, Albany, NY 12208-3479, USA; [email protected] (M.Z.); [email protected] (A.A.) * Correspondence: [email protected]

 Received: 10 December 2019; Accepted: 10 January 2020; Published: 15 January 2020 

Abstract: Chronic inflammation and subsequent tissue fibrosis are associated with a biochemical and mechanical remodeling of the fibronectin matrix. Due to its conformational lability, fibronectin is considerably stretched by the contractile forces of the fibrotic microenvironment, resulting in the unfolding of its Type III domains. In earlier studies, we have shown that a peptide mimetic of a partially unfolded fibronectin Type III domain, FnIII-1c, functions as a Damage Associated Molecular Pattern (DAMP) molecule to induce activation of a toll-like receptor 4 (TLR4)/NF-κB pathway and the subsequent release of fibro-inflammatory from human dermal fibroblasts. In the current study, we evaluated the requirement of the canonical TLR4/MD2/CD14 receptor complex in the regulation of FnIII-1c induced release. Using dermal fibroblasts and human embryonic kidney (HEK) cells, we found that all the components of the TLR4/MD2/CD14 complex were required for the release of the fibro-inflammatory cytokine, (IL-8) in response to both FnIII-1c and the canonical TLR4 ligand, (LPS). However, FnIII-1c mediated IL-8 release was strictly dependent on membrane-associated CD14, while LPS could use soluble CD14. These findings demonstrate that LPS and FnIII-1c share a similar but not identical mechanism of TLR4 activation in human dermal fibroblasts.

Keywords: fibronectin; TLR4; fibrosis; inflammation; IL-8; CD14

1. Introduction Chronic inflammation plays a significant role in many fibrotic diseases including cancer. Most solid tumors are characterized by an infiltration of fibroblasts, which under the influence of the tumor cells, differentiate into highly contractile myofibroblasts. The generation of the myofibroblast phenotype is accompanied by increases in both the fibronectin matrix and in the mechanical forces placed upon it. The signaling networks between stromal and cancer cells are exceedingly complex and interdependent, occurring on a background of poorly understood mechanical signals which are activated in response to increasing tissue rigidity. The is characterized by fibrosis and inflammation which contributes to tissue rigidity and is considered integral to tumor growth and [1]. Fibronectin is an (ECM) which is polymerized by adherent cells into a mechanically sensitive network of interacting fibers. Fibronectin is up-regulated in the stroma of solid tumors and has been shown to contribute to cancer cell growth, migration, invasion, survival and resistance to [2,3]. Consequently, the molecular pathways activated by stromal fibronectin are now regarded as potential drug targets [4]. However, the molecular pathways regulated by the pathological remodeling of stromal fibronectin are not well understood. Structurally, the fibronectin molecule consists of independently folded domains termed Type I, II, and III based on shared amino-acid homologies. Polymerized fibronectin fibers are conformationally labile and respond

Cells 2020, 9, 216; doi:10.3390/cells9010216 www.mdpi.com/journal/cells Cells 2020, 9, 216 2 of 13 to force by unfolding their Type III domains, which unlike the Type I and II domains, are not stabilized by disulfide bonds [5]. The unfolding of the Type III domains can cause fibronectin to stretch up to 8 times its length [6,7]. Studies have now demonstrated fibronectin in the stroma of solid tumors to be highly stretched due to the unfolding of Type III domains [8–10]. The impact of this strained form of fibronectin on cancer progression is not known. To understand the potential impact of fibronectin strain on tumor progression, we have used a fibronectin peptide, FnIII-1c, which corresponds to a stable intermediate structure predicted to form during force induced unfolding of the first Type III domain of fibronectin [11]. We have identified this peptide as a Damage Associated Molecular Pattern molecule or DAMP which induces the expression of several fibro-inflammatory in human dermal fibroblasts [12–14], DAMPs are endogenous products of tissue damage which work through toll-like receptors (TLR) to activate innate immune responses [15]. DAMPs arise early during tumor progression as the ECM is actively remodeled [16]. TLRs are a family of transmembrane receptors which were first identified on immune cells, as initiators of the innate immune response to pathogens, such as the bacterial cell wall component, LPS [17]. TLRs have also been identified on other cell types including fibroblasts, epithelial cells, endothelial cells and tumor cells [18–21]. TLRs function in complexes with co-receptors and ancillary whose specific functions are best understood for the activation of TLR4 by its prototype ligand, the Pathogen Associate Molecular Pattern molecule or PAMP, LPS [22]. TLR4 activation in response to LPS requires two accessory molecules, CD14 and MD2 [23]. MD2 is a secreted protein which complexes with TLR4, binds the moiety of LPS, and facilitates the formation of TLR4 dimers which are required for downstream signaling and activation of NF-κB[23]. CD14 is GPI-linked protein found on the cell membrane in lipid rafts and functions to transfer LPS from the bacterial cell wall to the MD2/TLR4 complex [24]. CD14 can also be lost from the cell surface by both protease dependent and independent shedding [25]. Shed CD14 is found in a soluble form in most body fluids including blood plasma and serum [26]. Soluble CD14 can also complex with LPS to initiate a TLR4 dependent inflammatory response [27]. FnIII-1c elicits expression of several fibro-inflammatory genes in dermal fibroblasts including IL-8 and -alpha (TNF-α). Cytokine release in response to FnIII-1c depends on a TLR4/NF-κB signaling pathway [12,13]. A variety of matrix-derived DAMPs have been identified in solid tumors including proteins, , and glycosaminoglycans. These DAMPs work primarily through a TLR4/NF-κB pathway to promote chronic inflammation and to drive the expression of growth and survival genes all of which are thought to contribute to tumor progression (reviewed in [28,29]). The molecular basis underlying the recognition of such a diverse array of ligands is not well understood. In some instances, TLRs can function in complexes with specific accessory proteins or co-receptors, thereby achieving ligand specific outcomes [30,31]. In the current study we evaluate the contribution of the components of the TLR4 receptor complex in the induction of cytokines by LPS and FnIII-1c. The data indicate that the fibronectin DAMP, FnIII-1c, similar to LPS, requires the TLR4/MD1/CD14 receptor complex to induce cytokine release from dermal fibroblasts. In contrast to LPS, FnIII-1c has a strict requirement for the membrane-bound form of CD14. These data suggest that targeting membrane-associated CD14 may provide a novel approach to designing therapeutics directed at controlling DAMP-dependent fibro-inflammatory disease.

2. Materials and Methods

2.1. Materials Ultrapure LPS purified from E. coli K12 strain was purchased from InvivoGen (San Diego, CA, USA). Human IL-8 enzyme-linked immunosorbent assay (ELISA) was from Becton Dickinson Biosciences (San Jose, CA, USA). Recombinant human CD14, human TNF-α, human IL-1α, anti-human MD-2 antibody, and neutralizing antibodies: anti-human CD14, anti-human TLR2 and anti-human TLR4 were purchased from R&D Systems (Minneapolis, MN, USA). DL-Sulforaphane and phosphatidylinositol Cells 2020, 9, 216 3 of 13 specific phospholipase C (PI-PLC) were purchased from Sigma-Aldrich-Millipore (St. Louis, MO, USA) TAK-242 was from Calbiochem/EMD Millipore (Billerica, MA, USA).

2.2. Recombinant Fibronectin Modules The recombinant fibronectin Type III modules were generated through polymerase chain reaction amplification of the human fibronectin cDNA clone pFH100 as previously described [32]. DNA encoding fibronectin amino acids Asn-631 to Pro-705 for III1c or Asn-1904 to Thr-1992 for III13 was cloned into vector pQE-70 (Qiagen, Germantown, MD, USA) and expressed in M15 bacteria. Recombinant proteins were purified by sequential chromatography with metal-chelating nitrilotriacetic acid agarose (Ni-NTA), sephadex G-25, and ion exchange columns as previously described [33].

2.3. Stably Transfected HEK 293 Cell Lines The pDUO-hTLR4A/MD2 (a pDUO plasmid co-expressing the human TLR4A and MD2 genes from InvivoGen) or pDUO-hTLR4A/CD14 plasmid (a pDUO plasmid co-expressing the human TLR4A and CD14 genes from InvivoGen (San Diego, CA, USA), were transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen/Life Technologies Corp., Grand Island, NY, USA). Two days later, cells were placed in selection medium (culture medium containing 10 µg/mL blasticidin). Clones were isolated from blasticidin-resistant cells by serial dilutions. Protein expression was screened by immunoblot analysis. A clone of 293-hTLR4A-MD2 and a clone of 293-hTLR4A-CD14 were selected for experiments. The other stably transfected HEK 293 cell lines: 293-Null, 293-hTLR4A, 293-hTLR4A-MD2-CD14, and 293-hMD2-CD14 were purchased from InvivoGen (San Diego, CA, USA).

2.4. Cell Culture Human dermal fibroblasts were grown in Dulbecco’s modified eagle medium (DMEM) (Invitrogen/Life Technologies, Corp., Grand Island, NY, USA) containing 50 U/mL penicillin, 50 mg/mL streptomycin, and 10% FBS (HyClone Laboratories/GE Healthcare Life Sciences, Logan, Utah, USA). 293-hTLR4-MD2-CD14 cells were maintained in DMEM containing 10 µg/mL blasticidin, 50 µg/mL of HygroGold, 50 U/mL penicillin, 50 mg/mL streptomycin and 100 µg/mL normocin. In addition, all other transfected HEK293 cells were maintained in DMEM containing 10 µg/mL blasticidin, 50 U/mL penicillin, 50 mg/mL streptomycin and 100 µg/mL normocin.

2.5. Cell Treatments Protease-free bovine serum albumin (BSA) from Roche Diagnostics (Indianapolis, IN, USA) was further processed for cell culture usage. Under sterile condition, 10% BSA in phosphate buffered saline (PBS) was dialyzed vs PBS containing 100 µg/mL Penicillin-Streptomycin (pen-strep) overnight with a dialysis tube (12–14 kDa molecular weight cut off) for 1 day and further vs DMEM containing pen-strep overnight. Dialyzed BSA solution’s volume was measured and the solution was sterilized with 0.22 µm syringe filter. BSA solution was inactivated by incubation at 56 ◦C for 1 h. For experiments, cells were placed in DMEM containing 10% FBS, 50 U/mL penicillin, 50 mg/mL streptomycin and 10 mM HEPES (10% FBS/DMEM), and DMEM containing 0.1% BSA, 50 U/mL penicillin, 50 mg/mL streptomycin, 1 non-essential amino acids and 10 mM HEPES (0.1% BSA/DMEM). × Human dermal fibroblasts were plated onto 48-well culture plates (2 104 cells per well) and cultured × overnight. When inhibitors, blocking antibodies or PI-PLC were used, cells were pre-incubated for the indicated times. For serum-free conditions, human dermal fibroblast monolayers were washed twice and incubated with 0.1% BSA/DMEM overnight prior to treatments. Transfected HEK293 cells were plated onto -coated 48-well culture plates (2 104 cells per well) and cultured for two days × in the presence of selection antibiotics. Cells were treated with fibronectin modules or LPS without selection antibiotics. In serum-free condition, transfected HEK293 monolayers were gently washed once and incubated with 0.1% BSA/DMEM for two hours prior to treatments. Cells 2020Cells, 92020, 216, 9, x FOR PEER REVIEW 4 of 134 of 13

HEK293 monolayers were gently washed once and incubated with 0.1% BSA/DMEM for two hours 2.6. Humanprior to IL-8treatments. Enzyme-Linked Immunosorbent Assay After cell treatment, condition medium was collected and centrifuged. Supernatant was saved 2.6. Human IL-8 Enzyme-Linked Immunosorbent Assay and properly diluted with PBS containing 10% FBS. IL-8 protein concentration in condition medium was determinedAfter cell by treatment, using a humancondition IL-8 medium ELISA was kit collected (BD Biosciences, and centrifuged. San Diego, Supernatant CA, USA). was saved and properly diluted with PBS containing 10% FBS. IL-8 protein concentration in condition medium 3. Resultswas determined by using a human IL-8 ELISA kit (BD Biosciences, San Diego, CA, USA).

3.1. Cytokine3. Results Induction in Response to Either FnIII-1c or LPS is Dependent on TLR4

To3.1. evaluateCytokine Induction the TLR in receptor Response complexto Either FnIII-1c on dermal or LPS fibroblasts is Dependent which on TLR4 regulates cytokine release in response to either pathogens or endogenous DAMPs, human dermal fibroblasts were incubated To evaluate the TLR receptor complex on dermal which regulates cytokine release in withresponse increasing to either amounts pathogens of either or endogenous the unfolded DAMP Types, III-1human domain dermal of fibrobla fibronectinsts were (FnIII-1c) incubated or with LPS. As we haveincreasing previously amounts reported of either [12 the,13 unfolded], incubation Type III- of1 human domain dermal of fibronectin fibroblasts (FnIII-1c) with or FnIII-1c LPS. As resultedwe in anhave increase previously in the reported synthesis [12,13], of IL-8 incubation (Figure1 ofA). human The e dermalffect of fibrob FnIII-1clasts onwith cytokine FnIII-1c expressionresulted in was dose-dependentan increase in reaching the synthesis over 30of IL ng-8/mL (Figure within 1A). 24 The h. Aeffect second of Fn fibronectinIII-1c on cytokine Type IIIexpression domain, was FnIII-13, did notdose-dependent elicit an IL-8 reaching response over and 30 ng/mL served within as negative 24 h. A second control. fibronectin LPS also Type induced III domain, a dose FnIII-13, response increasedid innot IL-8 elicit reaching an IL-8 overresponse 100 ngand/mL served within as 24negative h (Figure control.1B). InductionLPS also induced of IL-8 bya dose either response FnIII-1c or LPS wasincrease completely in IL-8 reaching inhibited over using 100 ng/mL a blocking withinantibody 24 h (Figure to 1B). TLR4. Induction Neither of controlIL-8 by either IgG norFnIII-1c blocking or LPS was completely inhibited using a blocking antibody to TLR4. Neither control IgG nor blocking antibody to TLR2 had any effect on IL-8 production in response to either FnIII-1c or LPS (Figure1C). antibody to TLR2 had any effect on IL-8 production in response to either FnIII-1c or LPS (Figure 1C). The inhibitor of downstream signaling from TLR4, TAK-242, also prevented the release of IL-8 in The inhibitor of downstream signaling from TLR4, TAK-242, also prevented the release of IL-8 in responseresponse to both to both FnIII-1c FnIII-1c and and LPS. LPS. In contrast,In contrast, TAK-242 TAK-242 had had only only a a slight slight eeffectffect onon thethe synthesis of of IL-8 in responseIL-8 in response to TNFα towhich TNFα doeswhich not does depend not depend on TLR4 on TLR4 signaling signaling (Figure (Figure1D). 1D). These These data dataindicate indicate that TLR4that signaling TLR4 signaling mediates mediates cytokine cytokine release release from dermalfrom dermal fibroblasts fibroblasts in response in response to both to both the bacterialthe pathogenbacterial LPS pathogen and the LPS matrix-derived and the matrix-derived DAMP, FnIII-1c. DAMP, FnIII-1c.

A 40 B 140 120 FnIII-1c 30 100 80 20 60 IL-8 (ng/ml) IL-8 10 (ng/ml) IL-8 40 20 0 FnIII-13 0 0 12 5 10 20 0310 30 100 (µg/mL) LPS (ng/mL)

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0 0 0.05 0.1 0.2 0.5 TAK-242 (µM) FigureFigure 1. TLR41. TLR4 mediates mediates IL-8 IL-8 expressionexpression in response response to to FnIII-1c FnIII-1c and and LPS LPS in dermal in dermal fibroblasts. fibroblasts. MonolayersMonolayers of human of human dermal dermal fibroblasts fibroblasts inin 10%10% FBS/DMEM FBS/DMEM were were treated treated for for24 h 24 with h with (A) FnIII-1c (A) FnIII-1c or FnIII-13or FnIII-13 (1-20 (1-20µg /µg/mL),mL), (B ()B LPS) LPS (1-100 (1-100 ngng/mL),/mL), ( CC)) LPS LPS (100 (100 ng/mL) ng/mL) or orFnIII-1c FnIII-1c (10 µM) (10 µinM) the in the presencepresence of the of designatedthe designated amounts amounts of of blocking blocking antibodyantibody to to TLR4 TLR4 or or TLR2. TLR2. IgG IgG served served as control. as control. (D) (D) TNF-α (25 ng/mL), LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of increasing amounts of the

TLR4 inhibitor, TAK-242. The wells without antibodies (C) or inhibitors (D) were set as 100%. IL-8 concentration in conditioned medium was determined by ELISA. The data represent the mean S.E. of ± triplicate assays from three separate experiments. Cells 2020, 9, x FOR PEER REVIEW 5 of 13

TNF-α (25 ng/mL), LPS (100 ng/mL) or FnIII-1c (10 µM) in the presence of increasing amounts of the TLR4 inhibitor, TAK-242. The wells without antibodies (C) or inhibitors (D) were set as 100%. IL-8 Cells 2020concentration, 9, 216 in conditioned medium was determined by ELISA. The data represent the mean ± S.E.5 of 13 of triplicate assays from three separate experiments. 3.2. TLR4 Signaling in Response to Either FnIII-1c or LPS Depends on MD2 and CD14 3.2. TLR4 Signaling in Response to Either FnIII-1c or LPS Depends on MD2 and CD14 Activation of TLR4 signaling on immune cells by LPS is known to depend on MD2. To determine Activation of TLR4 signaling on immune cells by LPS is known to depend on MD2. To determine whether MD2 is required for the activation of TLR4 signaling on dermal fibroblasts, cells were whether MD2 is required for the activation of TLR4 signaling on dermal fibroblasts, cells were pre- pre-incubated with sulforaphane, an inhibitor of MD2. As shown in Figure2A, sulforaphane inhibited incubated with sulforaphane, an inhibitor of MD2. As shown in Figure 2A, sulforaphane inhibited the synthesis of IL-8 in response to both LPS and FnIII-1c. In contrast, sulforaphane did not block IL-8 the synthesis of IL-8 in response to both LPS and FnIII-1c. In contrast, sulforaphane did not block IL- induction in response to TNF-α. 8 induction in response to TNF-α.

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Figure 2. IL-8 induction by LPS and FnIII-1c in dermal fibroblasts requires MD2 and CD14. Monolayers Figure 2. IL-8 induction by LPS and FnIII-1c in dermal fibroblasts requires MD2 and CD14. of human dermal fibroblasts were incubated with the (A) MD2 inhibitor, sulforaphane, in 10% Monolayers of human dermal fibroblasts were incubated with the (A) MD2 inhibitor, sulforaphane, FBS/DMEM or (B) CD14 blocking antibody or control normal IgG in 1% human serum/DMEM. Cells in 10% FBS/DMEM or (B) CD14 blocking antibody or control normal IgG in 1% human serum/DMEM. were then treated for 24 hours with either FnIII-1c (10 µM), LPS (100 ng/mL), or TNF-α (25 ng/mL) in Cells were then treated for 24 hours with either FnIII-1c (10 µM), LPS (100 ng/mL), or TNF-α (25 the continuing presence of antibodies or inhibitors. IL-8 concentration in conditioned medium was ng/mL) in the continuing presence of antibodies or inhibitors. IL-8 concentration in conditioned determined by ELISA. The wells without antibodies or inhibitors were set as control (100%). The data medium was determined by ELISA. The wells without antibodies or inhibitors were set as control represent the mean S.E. of triplicate assays from two separate experiments. (100%). The data represent± the mean ± S.E. of triplicate assays from two separate experiments. CD14 is a GPI-linked cell surface protein, typically found in lipid rafts in the plasma membrane whereCD14 it binds is a LPSGPI-linked and facilitates cell surface its transfer protein, to typically the TLR4 found/MD2 in complex lipid rafts to initiatein the plasma TLR signaling. membrane To whereevaluate it binds the role LPS of and CD14 facilitates in LPS andits transfer FnIII-1c to mediated the TLR4/MD2 cytokine complex release to by initiate dermal TLR fibroblasts, signaling. cells To wereevaluate incubated the role with of CD14 either in LPS LPS or and FnIII-1c FnIII-1c and increasingmediated cytokine concentrations release of by blocking dermal antibody fibroblasts, to CD14. cells wereUnder incubated these conditions, with either the stimulationLPS or FnIII-1c of IL-8 and production increasing by concentrations either LPS or of FnIII-1c blocking was antibody completely to CD14.inhibited Under by anti-CD14 these conditions, antibody. the Taken stimulation together, of these IL-8 data production indicate thatby either induction LPS ofor IL-8 FnIII-1c in dermal was completelyfibroblasts byinhibited both LPS by andanti-CD14 FnIII-1c antibody. requires activationTaken together, of the these TLR4 data/MD2 indicate/CD14 signalingthat induction complex. of IL- 8 in dermal fibroblasts by both LPS and FnIII-1c requires activation of the TLR4/MD2/CD14 signaling complex.3.3. IL-8 Induction by LPS Is Serum-Dependent in Dermal Fibroblasts In addition to its location on the plasma membrane, CD14 is also found in a soluble form in 3.3. IL-8 Induction by LPS is Serum-Dependent in Dermal Fibroblasts serum [34]. Both soluble and membrane CD14 have been reported to facilitate the activation of TLR4 by LPS. To evaluate the contribution of serum-derived CD14 in the release of cytokines by dermal fibroblasts, cells were incubated with LPS in the presence or absence of serum. Figure3A shows that in the absence of serum, fibroblasts were completely unresponsive to LPS, even at relatively high Cells 2020, 9, x FOR PEER REVIEW 6 of 13

In addition to its location on the plasma membrane, CD14 is also found in a soluble form in serum [34]. Both soluble and membrane CD14 have been reported to facilitate the activation of TLR4 byCells LPS.2020 , To9, 216 evaluate the contribution of serum-derived CD14 in the release of cytokines by dermal6 of 13 fibroblasts, cells were incubated with LPS in the presence or absence of serum. Figure 3A shows that in the absence of serum, fibroblasts were completely unresponsive to LPS, even at relatively high concentrations (1 µg/mL). In the presence of 10% serum, LPS elicited a dose-dependent increase in concentrations (1 µg/mL). In the presence of 10% serum, LPS elicited a dose-dependent increase in IL-8 release. In contrast to LPS, FnIII-1c stimulated a dose-dependent release of comparable levels of IL-8 release. In contrast to LPS, FnIII-1c stimulated a dose-dependent release of comparable levels of IL-8 in the presence or absence of serum (Figure3B). These results suggest that LPS but not FnIII-1c IL-8 in the presence or absence of serum (Figure 3B). These results suggest that LPS but not FnIII-1c has a serum requirement to induce synthesis of IL-8 in dermal fibroblasts. Taken together with the has a serum requirement to induce synthesis of IL-8 in dermal fibroblasts. Taken together with the observation that TLR4 signaling in response to LPS depends on CD14 (Figure2B), the data suggest observation that TLR4 signaling in response to LPS depends on CD14 (Figure 2B), the data suggest that the LPS-mediated synthesis of IL-8 by dermal fibroblasts depends on the soluble CD14 present in that the LPS-mediated synthesis of IL-8 by dermal fibroblasts depends on the soluble CD14 present serum. To confirm that soluble CD14 supports the induction of IL-8 by LPS, fibroblasts were incubated in serum. To confirm that soluble CD14 supports the induction of IL-8 by LPS, fibroblasts were with either LPS or FnIII-1c in the presence of exogenous purified soluble CD14. In the absence of incubated with either LPS or FnIII-1c in the presence of exogenous purified soluble CD14. In the exogenous CD14, fibroblasts synthesized very little IL-8 over a 24 hr period in response to LPS. In absence of exogenous CD14, fibroblasts synthesized very little IL-8 over a 24 hr period in response to contrast, treatment with Fn III-1c resulted in the release of over 20 ng/mL of IL-8 (Figure3C). When LPS. In contrast, treatment with Fn III-1c resulted in the release of over 20 ng/mL of IL-8 (Figure 3C). serum-free medium was supplemented with increasing concentrations of exogenous CD14, there When serum-free medium was supplemented with increasing concentrations of exogenous CD14, was a dose-dependent increase in IL-8 synthesis in response to both FnIII-1c and LPS. The data are there was a dose-dependent increase in IL-8 synthesis in response to both FnIII-1c and LPS. The data consistent with a role for CD14 in the initiation of TLR4 signaling in response to both LPS and FnIII-1c, are consistent with a role for CD14 in the initiation of TLR4 signaling in response to both LPS and but indicate that fibroblasts respond poorly to LPS unless supplemented with an exogenous source FnIII-1c, but indicate that fibroblasts respond poorly to LPS unless supplemented with an exogenous of CD14. In contrast, FnIII-1c elicited a robust cytokine response in the absence of exogenous CD14, source of CD14. In contrast, FnIII-1c elicited a robust cytokine response in the absence of exogenous CD14,indicating indicating that membrane-bound that membrane-bound CD14 is suCD14fficient is tosu initiatefficient TLR4to initiate signaling TLR4 in signaling dermal fibroblasts in dermal in fibroblastsresponse to in FnIII-1c. response to FnIII-1c.

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0 0 12 5 10 20 FnIII-1c (µM) C 60 LPS 50 FnIII-1c 40 30 20 IL-8 (ng/mL) 10 0 0131030 Soluble CD14 (ng/mL) Figure 3. Effect of serum and exogenous CD14 on LPS and FnIII-1c-induced IL-8 expression in dermal Figurefibroblasts. 3. Effect Monolayers of serum ofand human exogenous dermal CD14 fibroblasts on LPS were and FnIII-1c-induced incubated for 24 hoursIL-8 expression with the designated in dermal fibroblasts.concentrations Monolayers of (A) LPS of human or (B) FnIII-1cdermal fibroblasts in either 10% were FBS incubated/DMEM for or 24 0.1% hours BSA with/DMEM. the designated (C) Cells concentrationswere treated for of 24(A hours) LPS or with (B) LPS FnIII-1c (250 in ng /eithermL) or 10% FnIII-1c FBS/DMEM (10 µM) or in 0.1% the presenceBSA/DMEM. of increasing (C) Cells wereconcentrations treated for of 24 exogenous hours with soluble LPS (250 CD14 ng/mL) in 0.1% or BSAFnIII-1c/DMEM. (10 µM) IL-8 in concentration the presence in of conditioned increasing concentrationsmedium was measured of exogenous by ELISA. soluble The CD14 data in represent 0.1% BSA/DMEM. the mean IL-8S.E. ofconcentration triplicate assays in conditioned from three ± separate experiments.

Cells 2020, 9, x FOR PEER REVIEW 7 of 13

Cells 2020medium, 9, 216 was measured by ELISA. The data represent the mean ± S.E. of triplicate assays from three 7 of 13 separate experiments. 3.4. IL-8 Induction by FnIII-1c Requires Membrane-Bound CD14 in Dermal Fibroblasts 3.4. IL-8 Induction by FnIII-1c Requires Membrane-Bound CD14 in Dermal Fibroblasts The data indicate that dermal fibroblasts do not response to LPS unless provided with an The data indicate that dermal fibroblasts do not response to LPS unless provided with an exogenous source of CD14. In contrast, dermal fibroblasts do respond to FnIII-1c, suggesting that exogenous source of CD14. In contrast, dermal fibroblasts do respond to FnIII-1c, suggesting that membrane-bound CD14 is sufficient to activate TLR4 signaling in response to FnIII-1c but not LPS. membrane-bound CD14 is sufficient to activate TLR4 signaling in response to FnIII-1c but not LPS. CD14 is attached to the plasma membrane by a glycophosphatidylinositol (GPI) linkage and can be CD14 is attached to the plasma membrane by a glycophosphatidylinositol (GPI) linkage and can be removed from the membrane by treatment with the enzyme, Phosphatidylinositol-phospholipase removed from the membrane by treatment with the enzyme, Phosphatidylinositol-phospholipase C C (PI-PLC). To evaluate further the requirement for membrane-bound CD14 in mediating cytokine (PI-PLC). To evaluate further the requirement for membrane-bound CD14 in mediating cytokine release in response to FnIII-1c, dermal fibroblasts were pretreated with PI-PLC prior to incubation with release in response to FnIII-1c, dermal fibroblasts were pretreated with PI-PLC prior to incubation either FnIII-1c or LPS in the presence of serum. TNF and IL-1 treated cells served as a positive control with either FnIII-1c or LPS in the presence of serum. TNF and IL-1 treated cells served as a positive for IL-8 induction as the receptors for these molecules do not depend on CD14 for signaling. As shown control for IL-8 induction as the receptors for these molecules do not depend on CD14 for signaling. in Figure4A, pretreatment of cells with increasing amounts of PI-PLC resulted in a complete inhibition As shown in Figure 4A, pretreatment of cells with increasing amounts of PI-PLC resulted in a of IL-8 secretion in response to FnIII-1c, while having little effect on the response to LPS. Induction complete inhibition of IL-8 secretion in response to FnIII-1c, while having little effect on the response of IL-8 in response to either TNF or IL-1 was largely unaffected by enzyme treatment. The data are to LPS. Induction of IL-8 in response to either TNF or IL-1 was largely unaffected by enzyme consistent with data in Figure3 showing that the CD14 present in the serum is su fficient to activate treatment. The data are consistent with data in Figure 3 showing that the CD14 present in the serum TLR4 signaling in response to LPS. In contrast, enzyme treatment prevented activation of TLR4 in is sufficient to activate TLR4 signaling in response to LPS. In contrast, enzyme treatment prevented response to FnIII-1c consistent with FnIII-1c requiring membrane-bound CD14. activation of TLR4 in response to FnIII-1c consistent with FnIII-1c requiring membrane-bound CD14.

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50 IL-8 (ng/mL) IL-8 25

0

Figure 4. Effect of soluble and membrane CD14 on LPS and FnIII-1c-induced IL-8 expression. (A) FigureMonolayers 4. Effect of humanof soluble dermal and fibroblasts membrane were CD14 pre-incubated on LPS and for FnIII-1c-induced 90 min with various IL-8 concentrationsexpression. (A of) MonolayersPI-PLC in 10% of human FBS/DMEM. dermal Cells fibroblasts were then were treated pre- forincubated 4 hours for with 90 IL-1 minα with(50 ng various/mL), LPS concentrations (100 ng/mL), ofFnIII-1c PI-PLC (10 inµ 10%M), orFBS/DMEM. TNF-α (50 ngCells/mL) were in the then continuing treated for presence 4 hours of with PI-PLC IL-1 inα 10%(50 ng/mL), FBS/DMEM. LPS Cells(100 ng/mL),which were FnIII-1c not treated (10 µM), with or PI-PLC TNF- wereα (50 set ng/mL) as control in (100%)the continuing (B) HEK-293 presence cells engineered of PI-PLC to in express 10% FBS/DMEM.the designated Cells components which were of theTLR4not treated receptor with complexPI-PLC were were set incubated as control with (100%) LPS (1 (µBg) /HEK-293mL) or FnIII-1c cells engineered(20 µM) in 10%to express FBS-DMEM the designated for 24 hours. components Cells receiving of theTLR4 only receptor 10% FBS-DMEM complex servedwere incubated as control. with IL-8 LPSconcentration (1 µg/mL) inor conditioned FnIII-1c (20 mediumµM) in 10% was measuredFBS-DMEM by for ELISA. 24 hours. The data Cells represent receiving the only mean 10%S.E. FBS- of ± DMEMtriplicate served assays as from control. two IL-8 separate concentration experiments. in conditioned medium was measured by ELISA. The data represent the mean ± S.E. of triplicate assays from two separate experiments. The requirement for membrane-bound CD14 in TLR4 activation was also assessed using HEK-293 cells engineered to express various components of the TLR4/MD2/CD14 receptor complex. HEK-293 cells, which do not express any components of the TLR4 receptor complex, were transfected with

Cells 2020, 9, 216 8 of 13 Cells 2020, 9, x FOR PEER REVIEW 8 of 13 the genes forThe TLR4, requirement MD2, for or membrane-bound CD14 individually CD14 and in TLR4 in combination. activation was also The assessed cells expressing using HEK- various combinations293 cells of engineered receptor to components express various were componen then incubatedts of the TLR4/MD2/CD14 with either LPS receptor or FnIII-1c complex. in theHEK- presence 293 cells, which do not express any components of the TLR4 receptor complex, were transfected with of serum.the Asgenes shown for TLR4, in Figure MD2,4 B,or HEK-293CD14 individually cells expressing and in combination. only TLR4 The or cells TLR4-CD14 expressing did various not release IL-8 in responsecombinations to either of receptor LPS components or FnIII-1c, were confirming then incubated a requirement with either forLPS MD2or FnIII-1c in the in activationthe presence of TLR4 by theseof ligands. serum. As LPS shown was in ableFigure to 4B, induce HEK-293 IL-8 cells secretion expressing in only HEK-293 TLR4 or cells TLR4-CD14 expressing did not only release TLR4 and MD2. ThisIL-8 findingin response is consistent to either LPS with or FnIII-1c, an earlier confirmi observationng a requirement that LPS for does MD2 not in the require activation membrane-bound of TLR4 CD14 initiateby these TLR4 ligands. activation LPS was able (see to Figuresinduce IL-83A secretion and4A). in In HEK-293 contrast, cells FnIII-1c expressing did only not TLR4 elicit and an IL-8 MD2. This finding is consistent with an earlier observation that LPS does not require membrane- response under these conditions. However, in HEK-293 cells where CD14 was also expressed, FnIII-1c bound CD14 initiate TLR4 activation (see Figures 3A and 4A). In contrast, FnIII-1c did not elicit an did induceIL-8 IL-8response secretion, under consistentthese conditions. with theHowever, membrane in HEK-293 form cells of CD14 where being CD14 required was also forexpressed, the activation of TLR4FnIII-1c signaling did induce by FnIII-1c IL-8 secretion, (Figure consistent4B). with the membrane form of CD14 being required for the Treatmentactivation of of HEK-293TLR4 signalin cellsg by expressing FnIII-1c (Figure TLR4 4B)./MD2 or TLR4/MD2/CD14 with increasing amounts of LPS resultedTreatment in a robust of HEK-293 induction cells expr ofessing IL-8 in TLR4/MD2 both cell or types TLR4/MD2/C when treatmentsD14 with increasing were performed amounts in the presenceof of LPS 10% resulted serum in (Figurea robust5 A).induction In the of absence IL-8 in both of serum, cell types cells when lacking treatments CD14 were were performed non-responsive in to the presence of 10% serum (Figure 5A). In the absence of serum, cells lacking CD14 were non- LPS (Figure5B). In contrast, FnIII-1c was unable to elicit an IL-8 response in HEK-293 cells expressing responsive to LPS (Figure 5B). In contrast, FnIII-1c was unable to elicit an IL-8 response in HEK-293 TLR4/MD2cellsin expressing either the TLR4/MD2 presence in or either absence the presence of serum or (Figure absence5 ofC,D), serum suggesting (Figure 5C,D), that suggesting soluble CD14 that cannot supportsoluble TLR4 activationCD14 cannot by support FnIII-1c. TLR4 Consistent activation with by FnIII- this1c. prediction, Consistent incubationwith this prediction, of FnIII-1c incubation with HEK-293 cells expressingof FnIII-1c TLR4 with/ MD2HEK-293 did cells not ex inducepressing IL-8 TLR4/MD2 even when did supplementednot induce IL-8 even with when purified supplemented soluble CD14. In contrastwith LPS purified was able soluble to elicit CD14. an In IL-8 contrast response LPS was under able to these elicit conditionsan IL-8 response (Figure under5E). these These conditions experiments indicate(Figure that in 5E). dermal These fibroblasts experiments both indicate MD2 that and in CD14 dermal are fibroblasts required both for MD2 the TLR4 and CD14 dependent are required induction of for the TLR4 dependent induction of cytokines by either LPS or FnIII-1c. The data also show that cytokineswhile by eitherboth FnIII-1c LPS or and FnIII-1c. LPS require The data CD14 also for showTLR4 ac thattivation, while there both is FnIII-1c a strict requirement and LPS require for cell CD14 for TLR4 activation,surface membrane-bound there is a strict CD14 requirement in the activation for cell of surface TLR4 by membrane-bound FnIII-1c, while LPS CD14can use in the the soluble activation of TLR4 byform FnIII-1c, of CD14 while to initiate LPS cansignaling. use the soluble form of CD14 to initiate signaling.

A 10% Serum B 0.1% BSA 100 60 50 75 40 50 30 TLR4/MD2/CD14 20 TLR4/MD2/CD14 IL-8 (ng/mL) IL-8 IL-8 (ng/mL) IL-8 25 TLR4/MD2 TLR4/MD2 10 0 0 0 123 0 0.25 0.50 0.75 1.00 LPS (µg/mL) LPS (µg/mL)

C 30 10% Serum D 60 0.1% BSA 25 50 20 40 15 30 10 TLR4/MD2/CD14 20 TLR4/MD2/CD14 IL-8 (ng/mL) IL-8

IL-8 (ng/mL) IL-8 TLR4/MD2 TLR4/MD2 5 10 0 0 0 5101520 0 5 10 15 20 FnIII-1c (µM) FnIII-1c (µM) E 50

40 30 LPS (1µg/mL) 20 FnIII-1c (20 µM)

IL-8 (ng/mL) IL-8 10

0 0 0.25 0.50 0.75 1.00 Soluble CD14 (µg/mL)

Figure 5.FigureFnIII-1c-induced 5. FnIII-1c-induced IL-8 IL-8 expression expression requiresrequires membrane membrane CD14. CD14. HEK HEK cells cellsexpressing expressing either either TLR4/MD2TLR4/MD2 or TLR4 or/ MD2TLR4/MD2/CD14/CD14 were were incubated incubated for for 24 24 hours hours with with thethe designateddesignated concentrations concentrations of of LPS (A,B) orLPS FnIII-1c (A,B) or (C FnIII-1c,D) in either(C,D) in 10% either FBS 10%/DMEM FBS/DMEM (A,C ()A or,C) 0.1% or 0.1% BSA BSA/DMEM/DMEM ((B,D).). ( (EE) )HEK-293 HEK-293 cells expressingcells TLR4-MD2expressing TLR4-MD2 were treated were treated with 1withµg/ 1mL µg/mL LPS LPS or 20or 20µM µM FnIII-1c FnIII-1c in 0.1% 0.1% BSA/DMEM BSA/DMEM in in the

presence of the indicated concentration of exogenous soluble CD14 for 24 h. IL-8 concentration in the conditioned medium was measured by ELISA. The data represent the mean S.E. of triplicate assays ± from two (A–D) or three (E) separate experiments. Cells 2020, 9, 216 9 of 13

4. Discussion Sterile inflammation occurs when DAMPs are released into the extracellular microenvironment in response to injury or during the progression of fibro-inflammatory associated pathologies. DAMPs can promote by regulating the innate immune response during the inflammatory phase of wound repair [15]. DAMPs are recognized by TLR receptor complexes which activate the NF-κB dependent expression of inflammatory and profibrotic genes. When this process becomes dysregulated, feed forward loops are created which lead to chronic inflammation and eventual tissue fibrosis [35–37]. In the case of solid tumors, ECM-derived DAMPs are created when between the tumor and the stromal fibroblasts results in increased matrix proteolysis, changes in expression of ECM genes and exposure of cryptic sites within the matrix which arise from increased contractile force generated from myofibroblasts [28,38]. DAMPs are comprised of structurally diverse molecules which are derived from ECM as well as from damaged cells [39]. The molecular basis for the recognition of these DAMPs by TLRs remains largely unknown [40]. In the current study, we present a side-by-side comparison between the fibronectin-derived DAMP, FnIII-1c, which is known to activate TLR4 signaling [12,13] with the canonical TLR4 ligand, LPS. Our data indicate that both ligands induce the expression of the cytokine and IL-8 in dermal fibroblasts, and that IL-8 expression is prevented by inhibitors of TLR4, MD2, and CD14. These findings indicate that as with LPS, FnIII-1c induces IL-8 through the activation of the TLR4/MD2/CD14 complex. While this is not unexpected, it should be noted that not all ECM-derived DAMPs use CD14 or MD2 to activate TLR4. Hyaluronan is a large molecular weight glycosaminoglycan which is found in the ECM. Low molecular weight hyaluronan which is generated by hyaluronidase in response to injury binds the transmembrane protein, CD44 which then complexes with TLR4 in membrane rafts to activate downstream signaling [41]. Another ECM protein, Tenascin C is up-regulated following injury and during tumor progression. Tenascin C also serves as a TLR4 to promote expression of fibro-inflammatory genes. Activation of TLR4 signaling by Tenascin C does not require either MD2 or CD14 [42]. A recent study in has shown that the activation of the TLR4/NF-κB pathway by Tenascin C or LPS results in the generation of different phenotypes characterized by different subsets of activated signaling molecules and the expression of distinct genes [43]. These data suggest that the innate immune response is tailored to the specific DAMP or PAMP present in the tissue microenvironment. The molecular basis of this distinction is likely linked to the use of ligand specific TLR4 co-receptors and adaptor proteins which can discriminate among the various DAMPs present in the tissue. The present study documents a distinguishing characteristic of TLR4 activation by the fibronectin DAMP, FnIII-1c. FnIII-1c dependent IL8 secretion in dermal fibroblasts required the membrane-bound species of CD14. Removal of membrane-bound CD14 with phospholipase C, rendered the cells unresponsive to FnIII-1c, even in the presence of exogenous soluble CD14. In contrast, LPS could not induce IL-8 secretion in dermal fibroblasts unless supplemented with an exogenous source of CD14. The requirement for soluble CD14 to initiate TLR4 activation by LPS is not clear but may result from insufficient levels of membrane-bound CD14 on the surface of dermal fibroblasts. Previous studies have suggested that soluble CD14 may be required for TLR4 activation of CD-14 deficient cells such as endothelial or epithelial cells [44]. Nevertheless, FnIII-1c induced cytokine expression in dermal fibroblasts, suggesting there was sufficient CD14 to activate TLR4 in response to FnIII-1c. A similar finding was seen in HEK cells engineered to express TLR4/MD2 where LPS but not FnIII-1c could elicit an IL-8 response in the presence of serum while FnIII-1c induced IL-8 only when cells also expressed membrane-bound CD14. The inability of FnIII-1c to use soluble CD14 suggests that unlike LPS, FnIII-1c may not bind directly to CD14, but may require another as yet unidentified receptor which subsequently interacts with membrane-bound CD14. These data demonstrate that LPS and FnIII-1c activate TLR4 signaling by a mechanism that is only partially shared. The significance of the requirement for membrane-bound CD14 in the initiation of TLR4 signaling by FnIII-1c is not known. Such a requirement places an extra level of control on the immune response of fibroblasts, restricting Cells 2020, 9, 216 10 of 13 it to those cells expressing the appropriate co-receptor required for TLR4 activation or to those cells closely associated with strained fibronectin fibers. The conformational flexibility of fibronectin allows it to respond to changes in tissue stiffness and mechanical force within the tumor microenvironment. Fibronectin has several mechanically regulated activities including polymerization of soluble fibronectin [45,46] as well as the binding of [10], collagen [47], growth factors [48], cytokines [49] and bacterial adhesins [50]. The stromal tissue of squamous cell lung cancer is enriched for fibronectin, myofibroblasts, and IL8, supporting a model of myofibroblast generated contractile force placing fibronectin under sufficient strain to trigger the activation of TLR4 signaling [51]. In a recent study using a 3D model of tumor-associated ECM, we have shown that fibronectin in the tumor-associated ECM activates TLR4 signaling and IL-8 release in lung cancer cells (Cho et al., manuscript under review). The proposed activation of TLR4 and subsequent release of IL-8 in response to strained fibronectin present in the tumor stroma is significant. Many solid tumors show high levels of IL-8, a fibro-inflammatory cytokine, which promotes , tumor cell proliferation, metastasis and drug resistance [52–54]. Targeting IL-8 has been proposed as a treatment for several including [55,56], pancreatic [57], and pre-leukemic stem cells [58]. In patients with chronic obstructive pulmonary disease, IL-8 levels can be diagnostic of lung cancer [59] and serum IL-8 levels are correlative with lung cancer risk [60]. Understanding the molecular steps controlling the mechanically activated signals generated in the tumor ECM will help in the identification of a novel class of therapeutic targets which can be used in the treatment of drug resistant, metastatic tumors.

Author Contributions: M.Z. designed and performed experiments; A.A. performed experiments; P.J.M.-L. wrote manuscript. All authors have read and agreed to the published version of the manuscript. Funding: Support for this work was provided by National Institutes of Health (NIH) grant R21-AR0667956 and R01-CA58626 to P.J.M.-L. Conflicts of Interest: The authors declare no conflict of interest.

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