NF-κB mediates lipopolysaccharide-induced alternative pre-mRNA splicing of MyD88 in mouse macrophages.

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.

Nontuberculous Mycobacteria Show Differential Infectivity and Use Phospholipids to Antagonize LL-37.

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.

Comparison of pro- and anti-inflammatory responses in paired human primary airway epithelial cells and alveolar macrophages.

BACKGROUND: Airway epithelial cells and alveolar macrophages (AMs) are the first line of defense in the lung during infection. Toll-like receptor (TLR) agonists have been extensively used to define the regulation of inflammation in these cells. However, previous studies were performed in non-paired airway epithelial cells and AMs. The major goal of our study was to compare the pro- and anti-inflammatory responses of paired human primary airway epithelial cells and AMs to TLR3 and TLR4 agonists. METHODS: Tracheobronchial epithelial cells (TBEC) and AMs from four smokers and four non-smokers without lung disease were cultured with or without Poly(I:C) (PIC) (a TLR3 agonist) or LPS (a TLR4 agonist) for 4, 24 and 48 h. The immune responses of paired cells were compared. RESULTS: TBEC and AMs showed stronger pro-inflammatory cytokine (e.g., IL-8) responses to PIC and LPS, respectively. TLR3 and TLR4 mRNA levels were similar in non-stimulated TBEC and AMs. However, PIC stimulation in AMs led to sustained up-regulation of the immune negative regulators Tollip and A20, which may render AMs less sensitive to PIC stimulation than TBEC. Unlike AMs, TBEC did not increase NF-κB activation after LPS stimulation. Interestingly, smoking status was correlated with less TLR3 and IRAK-M expression in non-stimulated TBEC, but not in AMs. PIC-stimulated TBEC and LPS-stimulated AMs from smokers vs. non-smokers produced more IL-8. Finally, we show that expression of A20 and IRAK-M is strongly correlated in the two paired cell types. CONCLUSIONS: By using paired airway epithelial cells and AMs, this study reveals how these two critical types of lung cells respond to viral and bacterial pathogen associated molecular patterns, and provides rationale for modulating immune negative regulators to prevent excessive lung inflammation during respiratory infection.

Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.

Alternative pre-mRNA splicing of Toll-like receptor signaling components in peripheral blood mononuclear cells from patients with ARDS.

A key physiological feature of acute respiratory distress syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine whether splicing in TLR pathways is altered in patients with ARDS, we monitored alternative splicing of MyD88 and IRAK1, two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long proinflammatory mRNAs (MyD88L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 patients with ARDS and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in patients with ARDS in a proinflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in patients with ARDS. We conclude that pre-mRNA splicing of TLR signaling genes is altered in patients with ARDS, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches.

Limiting of the innate immune response by SF3A-dependent control of MyD88 alternative mRNA splicing.

Controlling infectious disease without inducing unwanted inflammatory disease requires proper regulation of the innate immune response. Thus, innate immunity needs to be activated when needed during an infection, but must be limited to prevent damage. To accomplish this, negative regulators of innate immunity limit the response. Here we investigate one such negative regulator encoded by an alternative splice form of MyD88. MyD88 mRNA exists in two alternative splice forms: MyD88L, a long form that encodes a protein that activates innate immunity by transducing Toll-like receptor (TLR) signals; and a short form that encodes a different protein, MyD88S, that inhibits the response. We find that MyD88S levels regulate the extent of inflammatory cytokine production in murine macrophages. MyD88S mRNA levels are regulated by the SF3A and SF3B mRNA splicing complexes, and these mRNA splicing complexes function with TLR signaling to regulate MyD88S production. Thus, the SF3A mRNA splicing complex controls production of a negative regulator of TLR signaling that limits the extent of innate immune activation.

An evolutionarily conserved innate immunity protein interaction network.

The innate immune response plays a critical role in fighting infection; however, innate immunity also can affect the pathogenesis of a variety of diseases, including sepsis, asthma, cancer, and atherosclerosis. To identify novel regulators of innate immunity, we performed comparative genomics RNA interference screens in the nematode Caenorhabditis elegans and mouse macrophages. These screens have uncovered many candidate regulators of the response to lipopolysaccharide (LPS), several of which interact physically in multiple species to form an innate immunity protein interaction network. This protein interaction network contains several proteins in the canonical LPS-responsive TLR4 pathway as well as many novel interacting proteins. Using RNAi and overexpression studies, we show that almost every gene in this network can modulate the innate immune response in mouse cell lines. We validate the importance of this network in innate immunity regulation in vivo using available mutants in C. elegans and mice.

Spatiotemporal inhibition of innate immunity signaling by the Tbc1d23 RAB-GAP.

We previously identified Tbc1d23 as a candidate novel regulator of innate immunity using comparative genomics RNA interference screens in Caenorhabditis elegans and mouse macrophages. Using Tbc1d23 knockout mice and macrophages engineered to overexpress Tbc1d23, we now show that Tbc1d23 is a general inhibitor of innate immunity signaling, strongly inhibiting multiple TLR and dectin-signaling pathways. Tbc1d23 likely acts downstream of the TLR-signaling adaptors MyD88 and Trif and upstream of the transcription factor XBP1. Importantly, like XBP1, Tbc1d23 affects the maintenance, but not the initiation, of inflammatory cytokine production induced by LPS. Tbc1d23 acts as a RAB-GAP to regulate innate immunity signaling. Thus, Tbc1d23 exerts its inhibitory effect on innate immunity signaling in a spatiotemporal fashion. The identification of a novel spatiotemporal regulator of innate immunity signaling validates the comparative genomics approach for innate immunity gene discovery.

RNA Binding Proteins that Mediate LPS-induced Alternative Splicing of the MyD88 Innate Immune Regulator.

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.

Thiol-Michael Addition Microparticles: Their Synthesis, Characterization, and Uptake by Macrophages.

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-associated U2AF1 mutations induce host defense defects by compromising neutrophil chemotaxis.

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.

Alternative pre-mRNA splicing as a mechanism for terminating Toll-like Receptor signaling.

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.

Mapping Macrophage Polarization and Origin during the Progression of the Foreign Body Response to a Poly(ethylene glycol) Hydrogel Implant.

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.

Novel regulators of the systemic response to lipopolysaccharide.

Our understanding of the role that host genetic factors play in the initiation and severity of infections caused by gram-negative bacteria is incomplete. To identify novel regulators of the host response to lipopolysaccharide (LPS), 11 inbred murine strains were challenged with LPS systemically. In addition to two strains lacking functional TLR4 (C3H/HeJ and C57BL/6J(TLR4-/-)), three murine strains with functional TLR4 (C57BL/6J, 129/SvImJ, and NZW/LacJ) were found to be relatively resistant to systemic LPS challenge; the other six strains were classified as sensitive. RNA from lung, liver, and spleen tissue was profiled on oligonucleotide microarrays to determine if unique transcripts differentiate susceptible and resistant strains. Gene expression analysis identified the Hedgehog signaling pathway and a number of transcription factors (TFs) involved in the response to LPS. RNA interference-mediated inhibition of six TFs (C/EBP, Cdx-2, E2F1, Hoxa4, Nhlh1, and Tead2) was found to diminish IL-6 and TNF-α production by murine macrophages. Mouse lines with targeted mutations were used to verify the involvement of two novel genes in innate immunity. Compared with wild-type control mice, mice deficient in the E2F1 transcription factor were found to have a reduced inflammatory response to systemic LPS, and mice heterozygote for Ptch, a gene involved in Hedgehog signaling, were found to be more responsive to systemic LPS. Our analysis of gene expression data identified novel pathways and transcription factors that regulate the host response to systemic LPS. Our results provide potential sepsis biomarkers and therapeutic targets that should be further investigated in human populations.

The Caenorhabditis elegans germ line regulates distinct signaling pathways to control lifespan and innate immunity.

The relationship between the mechanisms that control an organism's lifespan and its ability to respond to environmental challenges are poorly understood. In Caenorhabditis elegans, an insulin-like signaling pathway modulates lifespan and the innate immune response to bacterial pathogens via a common mechanism involving transcriptional regulation by the DAF-16/FOXO transcription factor. The C. elegans germ line also modulates lifespan in a daf-16-dependent manner. Here, we show that the germ line controls the innate immune response of C. elegans somatic cells to two different Gram-negative bacteria. In contrast to the insulin-like signaling pathway, the germ line acts via distinct signaling pathways to control lifespan and innate immunity. Under standard nematode culture conditions, the germ line regulates innate immunity in parallel to a known p38 MAPK signaling pathway, via a daf-16-independent pathway. Our findings indicate that a complex regulatory network integrates inputs from insulin-like signaling, p38 MAPK signaling, and germ line stem cells to control innate immunity in C. elegans. We also confirm that innate immunity and lifespan in C. elegans are distinct processes, as nonoverlapping regulatory networks control survival in the presence of pathogenic and nonpathogenic bacteria. Finally, we demonstrate that the p38 MAPK pathway in C. elegans is activated to a similar extent by both pathogenic and nonpathogenic bacteria, suggesting that both can induce the nematode innate immune response.

Identification of innate immunity genes and pathways using a comparative genomics approach.

To reveal regulators of innate immunity, we used RNAi assays to monitor the immune response when genes are inhibited in Caenorhabditis elegans and mouse macrophages. Genes that altered innate immune responsiveness in C. elegans were validated in murine macrophages, resulting in the discovery of 11 genes that regulate the innate immune response in both systems and the subsequent identification of a protein interaction network with a conserved role in innate immunity regulation. We confirmed the role of four of these 11 genes in antimicrobial gene regulation using available mutants in C. elegans. Several of these genes (acy-1, tub-2, and tbc-1) also regulate susceptibility to the pathogen Pseudomonas aeruginosa. These genes may prove critical to understanding host defense and represent potential therapeutic targets for infectious and immunological diseases.

MDS-associated SF3B1 mutations enhance proinflammatory gene expression in patient blast cells.

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.

Specificity and complexity of the Caenorhabditis elegans innate immune response.

In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an "antimicrobial fingerprint," which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.

Inflammation-Induced Alternative Pre-mRNA Splicing in Mouse Alveolar Macrophages.

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.