A Thy-1-negative immunofibroblast population emerges as a key determinant of fibrotic outcomes to biomaterials.

Fibrosis-associated fibroblasts have been identified across various fibrotic disorders, but not in the context of biomaterials, fibrotic encapsulation, and the foreign body response. In other fibrotic disorders, a fibroblast subpopulation defined by Thy-1 loss is strongly correlated with fibrosis yet we do not know what promotes Thy-1 loss. We have previously shown that Thy-1 is an integrin regulator enabling normal fibroblast mechanosensing, and here, leveraging nonfibrotic microporous annealed particle (MAP) hydrogels versus classical fibrotic bulk hydrogels, we demonstrate that Thy1-/- mice mount a fibrotic response to MAP gels that includes inflammatory signaling. We found that a distinct and cryptic α-smooth muscle actin-positive Thy-1- fibroblast population emerges in response to interleuklin-1ß (IL-1ß) and tumor necrosis factor-α (TNFα). Furthermore, IL-1ß/TNFα-induced Thy-1- fibroblasts consist of two distinct subpopulations that are strongly proinflammatory. These findings illustrate the emergence of a unique proinflammatory, profibrotic fibroblast subpopulation that is central to fibrotic encapsulation of biomaterials.

High-dimensional comparison of monocytes and T cells in post-COVID and idiopathic pulmonary fibrosis.

Introduction: Up to 30% of hospitalized COVID-19 patients experience persistent sequelae, including pulmonary fibrosis (PF). Methods: We examined COVID-19 survivors with impaired lung function and imaging worrisome for developing PF and found within six months, symptoms, restriction and PF improved in some (Early-Resolving COVID-PF), but persisted in others (Late-Resolving COVID-PF). To evaluate immune mechanisms associated with recovery versus persistent PF, we performed single-cell RNA-sequencing and multiplex immunostaining on peripheral blood mononuclear cells from patients with Early- and Late-Resolving COVID-PF and compared them to age-matched controls without respiratory disease. Results and discussion: Our analysis showed circulating monocytes were significantly reduced in Late-Resolving COVID-PF patients compared to Early-Resolving COVID-PF and non-diseased controls. Monocyte abundance correlated with pulmonary function forced vital capacity and diffusion capacity. Differential expression analysis revealed MHC-II class molecules were upregulated on the CD8 T cells of Late-Resolving COVID-PF patients but downregulated in monocytes. To determine whether these immune signatures resembled other interstitial lung diseases, we analyzed samples from Idiopathic Pulmonary Fibrosis (IPF) patients. IPF patients had a similar marked decrease in monocyte HLA-DR protein expression compared to Late-Resolving COVID-PF patients. Our findings indicate decreased circulating monocytes are associated with decreased lung function and uniquely distinguish Late-Resolving COVID-PF from Early-Resolving COVID-PF, IPF, and non-diseased controls.

Fluvastatin enhances IL-33-mediated mast cell IL-6 and TNF production.

Statins are HMG-CoA reductase inhibitors prescribed for lowering cholesterol. They can also inhibit inflammatory responses by suppressing isoprenylation of small G proteins. Consistent with this, we previously found that fluvastatin suppresses IgE-mediated mast cell function. However, some studies have found that statins induced pro-inflammatory cytokines in macrophages and NK cells. In contrast to IgE signaling, we show that fluvastatin augments IL-33-induced TNF and IL-6 production by mast cells. This effect required the key mast cell growth factor, stem cell factor (SCF). Treatment of IL-33-activated mast cells with mevalonic acid or isoprenoids reduced fluvastatin effects, suggesting fluvastatin acts at least partly by reducing isoprenoid production. Fluvastatin also enhanced IL-33-induced NF-κB transcriptional activity and promoted neutrophilic peritonitis in vivo, a response requiring mast cell activation. Other statins tested did not enhance IL-33 responsiveness. Therefore, this work supports observations of unexpected pro-inflammatory effects of some statins and suggests mechanisms by which this may occur. Because statins are candidates for repurposing in inflammatory disorders, our work emphasizes the importance of understanding the pleiotropic and possible unexpected effects of these drugs.

Lactate Is a Metabolic Mediator That Shapes Immune Cell Fate and Function.

Lactate and the associated H+ ions are still introduced in many biochemistry and general biology textbooks and courses as a metabolic by-product within fast or oxygen-independent glycolysis. However, the role of lactate as a fuel source has been well-appreciated in the field of physiology, and the role of lactate as a metabolic feedback regulator and distinct signaling molecule is beginning to gain traction in the field of immunology. We now know that while lactate and the associated H+ ions are generally immunosuppressive negative regulators, there are cell, receptor, mediator, and microenvironment-specific effects that augment T helper (Th)17, macrophage (M)2, tumor-associated macrophage, and neutrophil functions. Moreover, we are beginning to uncover how lactate and H+ utilize different transporters and signaling cascades in various immune cell types. These immunomodulatory effects may have a substantial impact in cancer, sepsis, autoimmunity, wound healing, and other immunomodulatory conditions with elevated lactate levels. In this article, we summarize the known effects of lactate and H+ on immune cells to hypothesize potential explanations for the divergent inflammatory vs. anti-inflammatory effects.

T. gondii infection induces IL-1R dependent chronic cachexia and perivascular fibrosis in the liver and skeletal muscle.

Cachexia is a progressive muscle wasting disease that contributes to death in a wide range of chronic diseases. Currently, the cachexia field lacks animal models that recapitulate the long-term kinetics of clinical disease, which would provide insight into the pathophysiology of chronic cachexia and a tool to test therapeutics for disease reversal. Toxoplasma gondii (T. gondii) is a protozoan parasite that uses conserved mechanisms to infect rodents and human hosts. Infection is lifelong and has been associated with chronic weight loss and muscle atrophy in mice. We have recently shown that T. gondii-induced muscle atrophy meets the clinical definition of cachexia. Here, the longevity of the T. gondii-induced chronic cachexia model revealed that cachectic mice develop perivascular fibrosis in major metabolic organs, including the adipose tissue, skeletal muscle, and liver by 9 weeks post-infection. Development of cachexia, as well as liver and skeletal muscle fibrosis, is dependent on intact signaling through the type I IL-1R receptor. IL-1α is sufficient to activate cultured fibroblasts and primary hepatic stellate cells (myofibroblast precursors in the liver) in vitro, and IL-1α is elevated in the sera and liver of cachectic, suggesting a mechanism by which chronic IL-1R signaling could be leading to cachexia-associated fibrosis.

Lactic Acid Inhibits Lipopolysaccharide-Induced Mast Cell Function by Limiting Glycolysis and ATP Availability.

Sepsis has a well-studied inflammatory phase, with a less-understood secondary immunosuppressive phase. Elevated blood lactate and slow lactate clearance are associated with mortality; however, regulatory roles are unknown. We hypothesized that lactic acid (LA) contributes to the late phase and is not solely a consequence of bacterial infection. No studies have examined LA effects in sepsis models in vivo or a mechanism by which it suppresses LPS-induced activation in vitro. Because mast cells can be activated systemically and contribute to sepsis, we examined LA effects on the mast cell response to LPS. LA significantly suppressed LPS-induced cytokine production and NF-κB transcriptional activity in mouse bone marrow-derived mast cells and cytokine production in peritoneal mast cells. Suppression was MCT-1 dependent and reproducible with sodium lactate or formic acid. Further, LA significantly suppressed cytokine induction following LPS-induced endotoxemia in mice. Because glycolysis is linked to inflammation and LA is a byproduct of this process, we examined changes in glucose metabolism. LA treatment reduced glucose uptake and lactate export during LPS stimulation. LA effects were mimicked by glycolytic inhibitors and reversed by increasing ATP availability. These results indicate that glycolytic suppression and ATP production are necessary and sufficient for LA effects. Our work suggests that enhancing glycolysis and ATP production could improve immune function, counteracting LA suppressive effects in the immunosuppressive phase of sepsis.

Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo.

Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.

Macrophage and Fibroblast Interactions in Biomaterial-Mediated Fibrosis.

Biomaterial-mediated inflammation and fibrosis remain a prominent challenge in designing materials to support tissue repair and regeneration. Despite the many biomaterial technologies that have been designed to evade or suppress inflammation (i.e., delivery of anti-inflammatory drugs, hydrophobic coatings, etc.), many materials are still subject to a foreign body response, resulting in encapsulation of dense, scar-like extracellular matrix. The primary cells involved in biomaterial-mediated fibrosis are macrophages, which modulate inflammation, and fibroblasts, which primarily lay down new extracellular matrix. While macrophages and fibroblasts are implicated in driving biomaterial-mediated fibrosis, the signaling pathways and spatiotemporal crosstalk between these cell types remain loosely defined. In this review, the role of M1 and M2 macrophages (and soluble cues) involved in the fibrous encapsulation of biomaterials in vivo is investigated, with additional focus on fibroblast and macrophage crosstalk in vitro along with in vitro models to study the foreign body response. Lastly, several strategies that have been used to specifically modulate macrophage and fibroblast behavior in vitro and in vivo to control biomaterial-mediated fibrosis are highlighted.

Polymer scaffold architecture is a key determinant in mast cell inflammatory and angiogenic responses.

Implanted polymer scaffolds can induce inflammation leading to the foreign body response (FBR), fibrosis, and implant failure. Thus, it is important to understand how immune cells interact with scaffolds to mitigate inflammation and promote a regenerative response. We previously demonstrated that macrophage phenotype is modulated by fiber and pore diameters of an electrospun scaffold. However, it is unclear if this effect is consistent among other innate immune cells. Mast cells are inflammatory sentinels that play a vital role in the FBR of implanted biomaterials, as well as angiogenesis. We determined if altering electrospun scaffold architecture modulates mast cell responses, with the goal of promoting regenerative cell-scaffold interactions. Polydioxanone (PDO) scaffolds were made from 60 mg/mL or 140 mg/mL PDO solutions, yielding structures with divergent fiber and pore diameters. Mouse mast cells plated on these scaffolds were activated with IL-33 or lipopolysaccharide (LPS). Relative to the 60 mg/mL scaffold, 140 mg/mL scaffolds yielded less IL-6 and TNF, and greater VEGF secretion. Pores >4-6 µm elicited less IL-6 and TNF secretion. IL-33-induced VEGF regulation was more complex, showing effects of both pore size and fiber diameter. These data indicate parameters that can predict mast cell responses to scaffolds, informing biomaterial design to increase wound healing and diminish implant rejection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 884-892, 2019.

TGF-ß1 Suppresses IL-33-Induced Mast Cell Function.

TGF-ß1 is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGF-ß1 can suppress IgE-mediated mast cell activation of human and mouse mast cells. IL-33 is a member of the IL-1 family capable of inducing mast cell responses and enhancing IgE-mediated activation. In this study, we investigated the effects of TGF-ß on IL-33-mediated mast cell activation. Bone marrow-derived mast cells cultured in TGF-ß1, ß2, or ß3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13, and MCP-1 in a concentration-dependent manner. TGF-ß1 inhibited IL-33-mediated Akt and ERK phosphorylation as well as NF-κB- and AP-1-mediated transcription. These effects were functionally important, as TGF-ß1 injection suppressed IL-33-induced systemic cytokines in vivo and inhibited IL-33-mediated cytokine release from human mast cells. TGF-ß1 also suppressed the combined effects of IL-33 and IgE-mediated activation on mouse and human mast cells. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGF-ß1 on IgE-mediated activation, demonstrate that TGF-ß1 can provide broad inhibitory signals to activated mast cells.

Galectin-1 promotes an M2 macrophage response to polydioxanone scaffolds.

Regulating soft tissue repair to prevent fibrosis and promote regeneration is central to creating a microenvironment conducive to soft tissue development. Macrophages play an important role in this process. The macrophage response can be modulated using biomaterials, altering cytokine and growth factor secretion to promote regeneration. Electrospun polydioxanone (PDO) fiber scaffolds promoted an M2 phenotype when macrophages were cultured on large diameter, highly porous scaffolds, but an M1 phenotype on smaller diameter fibers. In this study, we investigated whether incorporation of galectin-1, an immunosuppressive protein that enhances muscle regeneration, could promote the M2 response. Galectin-1 was incorporated into large and small fiber PDO scaffolds during electrospinning. Galectin-1 incorporation increased arginase-1 and reduced iNOS and IL-6 production in mouse bone-marrow derived macrophages compared with PDO alone for both scaffold types. Inhibition of ERK mitogen-activated protein kinase did not alter galectin-1 effects on arginase-1 and iNOS expression, but reversed IL-6 suppression, indicating that IL-6 is mediated by a different mechanism. Our results suggest that galectin-1 can be used to modulate macrophage commitment to a pro-regenerative M2 phenotype, which may positively impact tissue regeneration when using small diameter PDO scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2562-2571, 2017.

Lactic Acid Suppresses IL-33-Mediated Mast Cell Inflammatory Responses via Hypoxia-Inducible Factor-1α-Dependent miR-155 Suppression.

Lactic acid (LA) is present in tumors, asthma, and wound healing, environments with elevated IL-33 and mast cell infiltration. Although IL-33 is a potent mast cell activator, how LA affects IL-33-mediated mast cell function is unknown. To investigate this, mouse bone marrow-derived mast cells were cultured with or without LA and activated with IL-33. LA reduced IL-33-mediated cytokine and chemokine production. Using inhibitors for monocarboxylate transporters (MCT) or replacing LA with sodium lactate revealed that LA effects are MCT-1- and pH-dependent. LA selectively altered IL-33 signaling, suppressing TGF-ß-activated kinase-1, JNK, ERK, and NF-κB phosphorylation, but not p38 phosphorylation. LA effects in other contexts have been linked to hypoxia-inducible factor (HIF)-1α, which was enhanced in bone marrow-derived mast cells treated with LA. Because HIF-1α has been shown to regulate the microRNA miR-155 in other systems, LA effects on miR-155-5p and miR-155-3p species were measured. In fact, LA selectively suppressed miR-155-5p in an HIF-1α-dependent manner. Moreover, overexpressing miR-155-5p, but not miR-155-3p, abolished LA effects on IL-33-induced cytokine production. These in vitro effects of reducing cytokines were consistent in vivo, because LA injected i.p. into C57BL/6 mice suppressed IL-33-induced plasma cytokine levels. Lastly, IL-33 effects on primary human mast cells were suppressed by LA in an MCT-dependent manner. Our data demonstrate that LA, present in inflammatory and malignant microenvironments, can alter mast cell behavior to suppress inflammation.

Laminin- and basement membrane-polycaprolactone blend nanofibers as a scaffold for regenerative medicine.

Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.