Acute respiratory distress syndrome (ARDS) is characterized by robust inflammation in the lungs and systemic circulation. In this context, the toll-like receptor (TLR) signaling pathway plays a major role, driving inflammation that promotes host defense but also causing pathological tissue damage. To limit excessive inflammation, TLR signaling must be tightly controlled. One mechanism that modulates TLR signaling is alternative splicing of TLR pathway pre-mRNAs, which balances production of positively acting inflammatory mediators with alternative splice forms that terminate inflammation. To determine whether altered TLR pathway splicing contributes to pathological inflammation in ARDS, we evaluated two central mediators of the TLR signaling pathway, the MyD88 signaling adapter and the IRAK1 signaling kinase, in leukocytes isolated from bronchoalveolar lavage (BAL) of patients with ARDS. We found that MyD88 gene expression was decreased in BAL immune cells, whereas IRAK1 gene expression was increased. In parallel, we monitored long proinflammatory (MyD88-L and IRAK1) and shorter anti-inflammatory (MyD88-S and IRAK1c) splice forms and determined that IRAK1 splicing was shifted in a proinflammatory direction in patients with ARDS. Finally, we evaluated relationships between MyD88 isoform levels in BAL leukocytes and clinical outcomes. We conclude that pre-mRNA splicing of TLR pathway genes is altered in lung immune cells in patients with ARDS, that monitoring splicing of these genes may provide important prognostic information, and that manipulating splicing of these genes may be a useful novel therapeutic approach that needs further investigation.NEW & NOTEWORTHY We found that MyD88 expression is decreased, that IRAK1 expression is increased, and that IRAK1 splicing is shifted in a proinflammatory direction, in lung immune cells in patients with ARDS. We also find that MyD88 expression levels may correlate with survival in patients with ARDS. Thus, changes in expression and splicing of these two genes offer potential novel prognostic and therapeutic targets for ARDS.

Patients with myelodysplastic syndrome (MDS) are immunocompromised and are therefore susceptible to fatal infection. While neutropenia and neutrophil dysfunction account for much of this immunodeficiency, other immune cells likely also contribute. In contrast to the extensive study of neutrophils in MDS, there has been very little investigation of macrophage host defense function in MDS. In the current study, we find that macrophage differentiation and macrophage phagocytosis of bacteria are greatly weakened in patients with MDS, regardless of patient genotype. Moreover, we find that killing of those bacteria that are ingested is diminished in MDS patients with high-risk disease. Using a mouse model that expresses the MDS-associated U2AF1-S34F mutation, we find that this mutation is sufficient to induce macrophage functional defects. We conclude that macrophage host defense defects likely contribute to the immunodeficiency present in MDS.

Poly(ethylene glycol) hydrogels prepared from PEG diacrylate (PEGDA) monomers are widely investigated for biological applications including drug delivery and tissue engineering. Despite their high-water contents, PEGDA hydrogels when implanted into the body induce a foreign body response (FBR). The overall goal of this study was to investigate the role of surface adsorbed proteins in the FBR to PEGDA hydrogels and determine whether they act as damage associated molecular patterns (DAMPs) to initiate inflammation. Toll-like receptors (TLRs) 2 and 4 are one of the primary receptors that recognize DAMPs. In vitro and in vivo studies were performed using wildtype (Wt), TLR2-/-, TLR4-/-, and TLR2-/-TLR4-/- double knockout (DKO) mice. In vitro, Wt neutrophils were activated in response to the PEGDA hydrogels as measured by myeloperoxidase, and this response was partially mediated by TLR4 but not TLR2. Wt macrophages predisposed to an inflammatory state responded to the PEGDA hydrogel itself and to a greater extent to surface-adsorbed plasma by producing the pro-inflammatory cytokines interleukin 6 and tumor necrosis factor α. TLR4 and to a lesser extent TLR2 mediated this response. To link DAMPs to the FBR in vivo, PEGDA hydrogels were implanted subcutaneously in mice. The thickness of the inflammatory cell layer was mediated by both TLR2 and TLR4 as knocking out both TLRs led to significantly fewer inflammatory cells. The fibrous capsule was reduced by 50% in both single KO mice as well as the DKO mice. Taken together, this study determined that DAMPs formed from surface-adsorbed plasma activate TLR4. In the more complex in vivo environment, both TLR2 and TLR4 are major contributors to the inflammatory response and partial contributors to the fibrous encapsulation. Overall, these findings provide a critical link between DAMPs, TLRs, immune cells, and the FBR to PEGDA hydrogels.

The foreign body response (FBR) is a formidable reaction that occurs to any non-biological implantable biomaterial and results in fibrous encapsulation. Non-specific protein adsorption is the first stage of the FBR and is thought to initiate the response by activation of innate immune cells. Here we show that Toll-like receptors (TLRs) 2 and 4 are the primary receptors responsible for recognizing surface adsorbed proteins as damage associated molecular patterns (DAMPs) and they determine the material dependent FBR. An in vitro model using multiple biomaterials identified that macrophages, not neutrophils, respond to surface-adsorbed plasma via TLR2 and/or TLR4 and that deletion of both was required to inhibit activation across all materials. In the more complex in vivo environment, simultaneous deletion of TLR2 and TLR4 nearly abrogated the FBR to multiple biomaterials and eliminated the material dependencies in a subcutaneous implant mouse model. Deletion of either TLR2 or TLR4 showed either no effect or a partial reduction, depending on the material, demonstrating that TLRs determine the material-dependent FBR in vivo. Collectively, we identified TLR2 and TLR4 as necessary receptors for the FBR and implicate macrophage recognition of DAMPs of surface-adsorbed proteins, which vary depending on the material, as the main driver initiating the FBR. Our findings establish TLR2 and TLR4 as therapeutic targets to evade the FBR across a range of implantable materials. STATEMENT OF SIGNIFICANCE: Synthetic biomaterials when implanted elicit a foreign body response (FBR) leading to fibrous encapsulation. The mechanisms however are not fully understood. When a biomaterial is implanted, proteins non-specifically adsorb to the material. These proteins may act as damaged associated molecular patterns (DAMPs) to induce inflammation. Toll like receptor (TLR) 2 and 4 are known receptors that recognize DAMPs. This work investigated several different biomaterials and found that TLR2 and TLR4 mediate the FBR in a material-dependent manner. Deleting both TLR2 and TLR4 was necessary to inhibit significantly fibrous capsule formation across all materials tested. Our findings provide direct evidence that DAMPs are the main driver of the FBR and establish TLR2/4 as potential therapeutic targets to evade the FBR.

Inflammation driven by Toll-like receptor (TLR) signaling pathways is required to combat infection. However, inflammation can damage host tissues; thus it is essential that TLR signaling ultimately is terminated to prevent chronic inflammatory disorders. One mechanism that terminates persistent TLR signaling is alternative splicing of the MyD88 signaling adaptor, which functions in multiple TLR signaling pathways. While the canonical long isoform of MyD88 (MyD88-L) mediates TLR signaling and promotes inflammation, an alternatively-spliced shorter isoform of MyD88 (MyD88-S) produces a dominant negative inhibitor of TLR signaling. MyD88-S production is induced by inflammatory agonists including lipopolysaccharide (LPS), and thus MyD88-S induction is thought to act as a negative feedback loop that prevents chronic inflammation. Despite the potential role that MyD88-S production plays in inflammatory disorders, the mechanisms controlling MyD88 alternative splicing remain unclear. Here, we identify two RNA binding proteins, SRSF1 and HNRNPU, that regulate LPS-induced alternative splicing of MyD88.

Polymeric microparticles are promising biomaterial platforms for targeting macrophages in the treatment of disease. This study investigates microparticles formed by a thiol-Michael addition step-growth polymerization reaction with tunable physiochemical properties and their uptake by macrophages. The hexafunctional thiol monomer dipentaerythritol hexa-3-mercaptopropionate (DPHMP) and tetrafunctional acrylate monomer di(trimethylolpropane) tetraacrylate (DTPTA) were reacted in a stepwise dispersion polymerization, achieving tunable monodisperse particles over a size range (1-10 μm) relevant for targeting macrophages. An off-stoichiometry thiol-acrylate reaction afforded facile secondary chemical functionalization to create particles with different chemical moieties. Uptake of the microparticles by RAW 264.7 macrophages was highly dependent on treatment time, particle size, and particle chemistry with amide, carboxyl, and thiol terminal chemistries. The amide-terminated particles were non-inflammatory, while the carboxyl- and thiol-terminated particles induced pro-inflammatory cytokine production in conjunction with particle phagocytosis. Finally, a lung-specific application was explored through time-dependent uptake of amide-terminated particles by human alveolar macrophages in vitro and mouse lungs in vivo without inducing inflammation. The findings demonstrate a promising microparticulate delivery vehicle that is cyto-compatible, is non-inflammatory, and exhibits high rates of uptake by macrophages.

Myelodysplastic neoplasm (MDS) is a hematopoietic stem cell disorder that may evolve into acute myeloid leukemia. Fatal infection is among the most common cause of death in MDS patients, likely due to myeloid cell cytopenia and dysfunction in these patients. Mutations in genes that encode components of the spliceosome represent the most common class of somatically acquired mutations in MDS patients. To determine the molecular underpinnings of the host defense defects in MDS patients, we investigated the MDS-associated spliceosome mutation U2AF1-S34F using a transgenic mouse model that expresses this mutant gene. We found that U2AF1-S34F causes a profound host defense defect in these mice, likely by inducing a significant neutrophil chemotaxis defect. Studies in human neutrophils suggest that this effect of U2AF1-S34F likely extends to MDS patients as well. RNA-seq analysis suggests that the expression of multiple genes that mediate cell migration are affected by this spliceosome mutation and therefore are likely drivers of this neutrophil dysfunction.

While inflammation induced by Toll-like receptor (TLR) signaling is required to combat infection, persistent inflammation can damage host tissues and contribute to a myriad of acute and chronic inflammatory disorders. Thus, it is essential not only that TLR signaling be activated in the presence of pathogens but that TLR signaling is ultimately terminated. One mechanism that limits persistent TLR signaling is alternative pre-mRNA splicing. In addition to encoding the canonical mRNAs that produce proteins that promote inflammation, many genes in the TLR signaling pathway also encode alternative mRNAs that produce proteins that are dominant negative inhibitors of signaling. Many of these negative regulators are induced by immune challenge, so production of these alternative isoforms represents a negative feedback loop that limits persistent inflammation. While these alternative splicing events have been investigated on a gene by gene basis, there has been limited systemic analysis of this mechanism that terminates TLR signaling. Here we review what is known about the production of negatively acting alternative isoforms in the TLR signaling pathway including how these inhibitors function, how they are produced, and what role they may play in inflammatory disease.

Poly(ethylene glycol) (PEG) hydrogels hold promise for in vivo applications but induce a foreign body response (FBR). While macrophages are key in the FBR, many questions remain. This study investigates temporal changes in the transcriptome of implant-associated monocytes and macrophages. Proinflammatory pathways are upregulated in monocytes compared to control monocytes but subside by day 28. Macrophages are initially proinflammatory but shift to a profibrotic state by day 14, coinciding with fibrous capsule emergence. Next, this study assesses the origin of macrophages responsible for fibrous encapsulation using wildtype, C-C Motif Chemokine Receptor 2 (CCR2)-/- mice that lack recruited macrophages, and Macrophage Fas-Induced Apoptosis (MaFIA) mice that enable macrophage ablation. Subpopulations of recruited and tissue-resident macrophages are identified. Fibrous encapsulation proceeds in CCR2-/- mice similar to wildtype mice. However, studies in MaFIA mice indicate that macrophages are necessary for fibrous capsule formation. These findings suggest that macrophage origin impacts the FBR progression and provides evidence that tissue-resident macrophages and not the recruited macrophages may drive fibrosis in the FBR to PEG hydrogels. This study demonstrates that implant-associated monocytes and macrophages have temporally distinct transcriptomes in the FBR and that profibrotic pathways associated with macrophages may be enriched in tissue-resident macrophages.

Background: The mechanisms that cause the progression of myelodysplastic syndrome (MDS) are poorly understood. Little is known about major signaling networks and energy metabolism in MDS cells as patients progress from low risk (LR) to high risk (HR) disease and from high risk to secondary acute myelogenous leukemia (sAML). As many as 30% of HR MDS patients progress to sAML and a portion of LR MDS patients progress to HR. The goal of this project is preventing progression by identifying MDS-specific targets for therapy. A deeper understanding of the metabolic properties of leukemia stem cells (LSCs) in AML has shown these cells are uniquely vulnerable to venetoclax and azacitidine (Ven/Aza) (Pollyea et al, Nat. Medicine, 2018) and metabolic changes cause resistance to Ven/Aza (Stevens et al, Nat. Cancer, 2021). Little however is known about the contribution of metabolism to the pathogenesis of MDS. The contributing factors to progression including metabolic properties, transcriptional programs, and immunophenotype are examined in this study.

Synthetic hydrogels, such as poly(ethylene glycol) (PEG), are promising for a range of in vivo applications. However, like all non-biological biomaterials, synthetic hydrogels including PEG elicit a foreign body response (FBR). The FBR is thought to be initiated by adsorbed protein that is recognized by and subsequently activates inflammatory cells, notably macrophages, and culminates with fibrotic encapsulation. However, the molecular mechanisms that drive the FBR are not well understood. Toll-like receptors (TLRs) are key receptors that recognize pathogens, but also recognize altered host proteins that display damage-associated molecular patterns (DAMPs). Thus TLRs may play a role in the FBR. Here, we investigated myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor protein that mediates inflammatory cytokine production induced by most TLRs. An in vitro model was used consisting of macrophages cultured on the surface of synthetic hydrogels, specifically PEG, with pre-adsorbed serum proteins. Our in vitro findings demonstrate that MyD88-dependent signaling is the predominant inflammatory pathway in macrophage activation to synthetic hydrogels. When stimulated with TLR agonists to mimic additional DAMPs present in vivo, MyD88-dependent signaling was also the predominant pathway in macrophage activation. An in vivo model of PEG hydrogels implanted subcutaneously in wild-type and MyD88 −/− mice also demonstrated that MyD88 is the key contributor to the recruitment of inflammatory cells and formation of the fibrous capsule surrounding the implanted hydrogel. Taken together, findings from this study identify MyD88-mediated inflammation as being a critical pathway involved not only in the inflammatory response, but in formation of the fibrous capsule to PEG hydrogels. underlying this response remain to be elucidated. Synthetic hydrogels are promising for in vivo applications but, like all non-biological biomaterials, synthetic hydrogels elicit a foreign body response (FBR). The molecular mechanisms that drive the FBR are not well understood. This work identifies the myeloid differentiation primary response gene 88 (MyD88) as a central mediator to macrophage activation in response to a poly(ethylene glycol) hydrogel with pre-adsorbed proteins in vitro. Moreover, MyD88 was also central to the recruitment of inflammatory cells, which included neutrophils, monocytes, and macrophages, to implanted PEG hydrogels and to fibrous encapsulation. These findings demonstrate that MyD88-mediated inflammation is responsible in part for the formation of the fibrous capsule of the FBR.

Frank Fang-Yao Lee, Kevin Davidson , X Chelsea Harris, Jazalle McClendon**, X William J. Janssen**, and Scott Alper From the Departments of Biomedical Research and **Medicine and the Center for Genes, Environment, and Health, National Jewish Health, Denver, Colorado 80206, the Department of Immunology andMicrobiology and the Division of Pulmonary Sciences and Critical CareMedicine, Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado 80045, and Pulmonary and Critical Care, WakeMed Hospital, Raleigh, North Carolina 27610

Two factors known to contribute to the development of myelodysplastic syndrome (MDS) and other blood cancers are (i) somatically acquired mutations in components of the spliceosome and (ii) increased inflammation. Spliceosome genes, including SF3B1, are mutated at high frequency in MDS and other blood cancers; these mutations are thought to be neomorphic or gain-of-function mutations that drive disease pathogenesis. Likewise, increased inflammation is thought to contribute to MDS pathogenesis; inflammatory cytokines are strongly elevated in these patients, with higher levels correlating with worsened patient outcome. In the current study, we used RNAseq to analyze pre-mRNA splicing and gene expression changes present in blast cells isolated from MDS patients with or without SF3B1 mutations. We determined that SF3B1 mutations lead to enhanced proinflammatory gene expression in these cells. Thus, these studies suggest that SF3B1 mutations could contribute to MDS pathogenesis by enhancing the proinflammatory milieu in these patients.

Although a robust inflammatory response is needed to combat infection, this response must ultimately be terminated to prevent chronic inflammation. One mechanism that terminates inflammatory signaling is the production of alternative mRNA splice forms in the Toll-like receptor (TLR) signaling pathway. Whereas most genes in the TLR pathway encode positive mediators of inflammatory signaling, several, including that encoding the MyD88 signaling adaptor, also produce alternative spliced mRNA isoforms that encode dominant-negative inhibitors of the response. Production of these negatively acting alternatively spliced isoforms is induced by stimulation with the TLR4 agonist lipopolysaccharide (LPS); thus, this alternative pre-mRNA splicing represents a negative feedback loop that terminates TLR signaling and prevents chronic inflammation. In the current study, we investigated the mechanisms regulating the LPS-induced alternative pre-mRNA splicing of the MyD88 transcript in murine macrophages. We found that 1) the induction of the alternatively spliced MyD88 form is due to alternative pre-mRNA splicing and not caused by another RNA regulatory mechanism, 2) MyD88 splicing is regulated by both the MyD88- and TRIF-dependent arms of the TLR signaling pathway, 3) MyD88 splicing is regulated by the NF-κB transcription factor, and 4) NF-κB likely regulates MyD88 alternative pre-mRNA splicing per se rather than regulating splicing indirectly by altering MyD88 transcription. We conclude that alternative splicing of MyD88 may provide a sensitive mechanism that ensures robust termination of inflammation for tissue repair and restoration of normal tissue homeostasis once an infection is controlled.

Alveolar macrophages serve as central orchestrators of inflammatory responses in the lungs, both initiating their onset and promoting their resolution. However, the mechanisms that program macrophages for these dynamic responses are not fully understood. Over 95% of all mammalian genes undergo alternative pre-mRNA splicing. While alternative splicing has been shown to regulate inflammatory responses in macrophages in vitro, it has not been investigated on a genome-wide scale in vivo. Here we used RNAseq to investigate alternative pre-mRNA splicing in alveolar macrophages isolated from lipopolysaccharide (LPS)-treated mice during the peak of inflammation and during its resolution. We found that lung inflammation induced substantial alternative pre-mRNA splicing in alveolar macrophages. The number of changes in isoform usage was greatest at the peak of inflammation and involved multiple classes of alternative pre-mRNA splicing events. Comparative pathway analysis of inflammation-induced changes in alternative pre-mRNA splicing and differential gene expression revealed overlap of pathways enriched for immune responses such as chemokine signaling and cellular metabolism. Moreover, alternative pre-mRNA splicing of genes in metabolic pathways differed in tissue resident vs. recruited (blood monocyte-derived) alveolar macrophages and corresponded to changes in core metabolism, including a switch to Warburg-like metabolism in recruited macrophages with increased glycolysis and decreased flux through the tricarboxylic acid cycle.

Comparisons of infectivity among the clinically important nontuberculous mycobacteria (NTM) species have not been explored in great depth. Rapid-growing mycobacteria, including Mycobacterium abscessus and M. porcinum, can cause indolent but progressive lung disease. Slow-growing members of the M. avium complex are the most common group of NTM to cause lung disease, and molecular approaches can now distinguish between several distinct species of M. avium complex including M. intracellulare, M. avium, M. marseillense, and M. chimaera. Differential infectivity among these NTM species may, in part, account for differences in clinical outcomes and response to treatment; thus, knowing the relative infectivity of particular isolates could increase prognostication accuracy and enhance personalized treatment. Using human macrophages, we investigated the infectivity and virulence of nine NTM species, as well as multiple isolates of the same species. We also assessed their capacity to evade killing by the antibacterial peptide cathelicidin (LL-37). We discovered that the ability of different NTM species to infect macrophages varied among the species and among isolates of the same species. Our biochemical assays implicate modified phospholipids, which may include a phosphatidylinositol or cardiolipin backbone, as candidate antagonists of LL-37 antibacterial activity. The high variation in infectivity and virulence of NTM strains suggests that more detailed microbiological and biochemical characterizations are necessary to increase our knowledge of NTM pathogenesis.

Synthetic hydrogels, such as poly(ethylene glycol) (PEG), are promising for a range of in vivo applications. However, like all non-biological biomaterials, synthetic hydrogels including PEG elicit a foreign body response (FBR). The FBR is thought to be initiated by adsorbed protein that is recognized by and subsequently activates inflammatory cells, notably macrophages, and culminates with fibrotic encapsulation. However, the molecular mechanisms that drive the FBR are not well understood. Toll-like receptors (TLRs) are key receptors that recognize pathogens, but also recognize altered host proteins that display damage-associated molecular patterns (DAMPS). Thus TLRs may play a role in the FBR. Here, we investigated myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor protein that mediates inflammatory cytokine production induced by most TLRs. An in vitro model was used consisting of macrophages cultured on the surface of synthetic hydrogels, specifically PEG, with pre-adsorbed serum proteins. Our in vitro findings demonstrate that MyD88-dependent signaling is the predominant inflammatory pathway in macrophage activation to synthetic hydrogels. When stimulated with TLR agonists to mimic additional DAMPs present in vivo, MyD88-dependent signaling was also the predominant pathway in macrophage activation. An in vivo model of PEG hydrogels implanted subcutaneously in wild-type and MyD88(-/-) mice also demonstrated that MyD88 is the key contributor to the recruitment of inflammatory cells and formation of the fibrous capsule surrounding the implanted hydrogel. Taken together, findings from this study identify MyD88-mediated inflammation as being a critical pathway involved not only in the inflammatory response, but in formation of the fibrous capsule to PEG hydrogels. Statement of Significance Synthetic hydrogels are promising for in vivo applications but, like all non-biological biomaterials, synthetic hydrogels elicit a foreign body response (FBR). The molecular mechanisms that drive the FBR are not well understood. This work identifies the myeloid differentiation primary response gene 88 (MyD88) as a central mediator to macrophage activation in response to a poly(ethylene glycol) hydrogel with pre-adsorbed proteins in vitro. Moreover, MyD88 was also central to the recruitment of inflammatory cells, which included neutrophils, monocytes, and macrophages, to implanted PEG hydrogels and to fibrous encapsulation. These findings demonstrate that MyD88-mediated inflammation is responsible in part for the formation of the fibrous capsule of the FBR. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Genes encoding spliceosome components including SF3B1, U2AF1 , and SRSF2 , are frequently somatically mutated in myelodysplastic syndromes (MDS), other hematologic malignancies, and solid tumors.[1][1] Typically these are mutually exclusive, heterozygous, missense, hotspot mutations that result in

In this chapter, we describe methods for functional genomics studies in mouse macrophages. In particular, we describe complementary methods for gene inhibition using RNA interference (RNAi) and gene overexpression. These methods are readily amenable to medium- and high-throughput functional genomics investigations. These complementary loss-of-function and gain-of-function genomic approaches provide a rapid means of investigating the function of candidate genes prior to initiating more cumbersome studies in vivo.

Myelodysplastic syndrome (MDS) is a malignant hematopoietic stem cell disorder that frequently evolves into acute myeloid leukemia (AML). Patients with MDS are prone to infectious complications, in part due to the presence of severe neutropenia and/or neutrophil dysfunction. However, not all patients with neutropenia become infected, suggesting that other immune cells may compensate in these patients. Monocytes are also integral to immunologic defense; however, much less is known about monocyte function in patients with MDS. In the current study, we monitor the composition of peripheral blood monocytes and several aspects of monocyte function in MDS patients, including HLA-DR expression, LPS-induced inflammatory cytokine production, and phagocytosis. We find that monocytes from MDS patients exhibit relatively normal innate immune functions compared to monocytes from healthy control subjects. We also find that HLA-DR expression is moderately increased in monocytes from MDS patients. These results suggest that monocytes could compensate for other immune deficits in MDS patients to help fight infection. We also find that the range of immune functions in monocytes from MDS patients correlates with several key clinical parameters, including blast cell count, monocyte count, and revised International Prognostic Scoring System score, suggesting that disease severity impacts monocyte function in MDS patients.