Alveolar epithelial cells of lung fibrosis patients are susceptible to severe virus-induced injury.

Patients with pulmonary fibrosis (PF) often experience exacerbations of their disease, characterised by a rapid, severe deterioration in lung function that is associated with high mortality. Whilst the pathobiology of such exacerbations is poorly understood, virus infection is a trigger. The present study investigated virus-induced injury responses of alveolar and bronchial epithelial cells (AECs and BECs, respectively) from patients with PF and age-matched controls (Ctrls). Air-liquid interface (ALI) cultures of AECs, comprising type I and II pneumocytes or BECs were inoculated with influenza A virus (H1N1) at 0.1 multiplicity of infection (MOI). Levels of interleukin-6 (IL-6), IL-36γ and IL-1ß were elevated in cultures of AECs from PF patients (PF-AECs, n = 8-11), being markedly higher than Ctrl-AECs (n = 5-6), 48 h post inoculation (pi) (P<0.05); despite no difference in H1N1 RNA copy numbers 24 h pi. Furthermore, the virus-induced inflammatory responses of PF-AECs were greater than BECs (from either PF patients or controls), even though viral loads in the BECs were overall 2- to 3-fold higher than AECs. Baseline levels of the senescence and DNA damage markers, nuclear p21, p16 and H2AXγ were also significantly higher in PF-AECs than Ctrl-AECs and further elevated post-infection. Senescence induction using etoposide augmented virus-induced injuries in AECs (but not viral load), whereas selected senotherapeutics (rapamycin and mitoTEMPO) were protective. The present study provides evidence that senescence increases the susceptibility of AECs from PF patients to severe virus-induced injury and suggests targeting senescence may provide an alternative option to prevent or treat the exacerbations that worsen the underlying disease.

Three dimensional fibrotic extracellular matrix directs microenvironment fiber remodeling by fibroblasts.

Idiopathic pulmonary fibrosis (IPF), for which effective treatments are limited, results in excessive and disorganized deposition of aberrant extracellular matrix (ECM). An altered ECM microenvironment is postulated to contribute to disease progression through inducing profibrotic behavior of lung fibroblasts, the main producers and regulators of ECM. Here, we examined this hypothesis in a 3D in vitro model system by growing primary human lung fibroblasts in ECM-derived hydrogels from non-fibrotic (control) or IPF lung tissue. Using this model, we compared how control and IPF lung-derived fibroblasts responded in control and fibrotic microenvironments in a combinatorial manner. Culture of fibroblasts in fibrotic hydrogels did not alter in the overall amount of collagen or glycosaminoglycans but did cause a drastic change in fiber organization compared to culture in control hydrogels. High-density collagen percentage was increased by control fibroblasts in IPF hydrogels at day 7, but decreased at day 14. In contrast, IPF fibroblasts only decreased the high-density collagen percentage at day 14, which was accompanied by enhanced fiber alignment in IPF hydrogels. Similarly, stiffness of fibrotic hydrogels was increased only by control fibroblasts by day 14 while those of control hydrogels were not altered by fibroblasts. These data highlight how the ECM-remodeling responses of fibroblasts are influenced by the origin of both the cells and the ECM. Moreover, by showing how the 3D microenvironment plays a crucial role in directing cells, our study paves the way in guiding future investigations examining fibrotic processes with respect to ECM remodeling responses of fibroblasts. STATEMENT OF SIGNIFICANCE: In this study, we investigated the influence of the altered extracellular matrix (ECM) in Idiopathic Pulmonary Fibrosis (IPF), using a 3D in vitro model system composed of ECM-derived hydrogels from both IPF and control lungs, seeded with human IPF and control lung fibroblasts. While our results indicated that fibrotic microenvironment did not change the overall collagen or glycosaminoglycan content, it resulted in a dramatically alteration of fiber organization and mechanical properties. Control fibroblasts responded differently from IPF fibroblasts, highlighting the unique instructive role of the fibrotic ECM and the interplay with fibroblast origin. These results underscore the importance of 3D microenvironments in guiding pro-fibrotic responses, offering potential insights for future IPF therapies as well as other fibrotic diseases and cancer.

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition.

BACKGROUND: We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice. METHODS: WT, IL1KO or TNFRKO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1ß (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs. RESULTS: In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1KO and DSS-PN TNFRKO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1KO and DSS-PN TNFRKO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1KO and TNFRKO. Intraperitoneal injection of IL-1ß or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2. CONCLUSIONS: Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1ß and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation.

Application of human iPSC-derived macrophages in a miniaturized high-content-imaging-based efferocytosis assay.

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages. These IDM cells resembled MDMs both with respect to surface marker expression and phago- as well as efferocytotic function. A statistically robust high-content-imaging assay was developed to quantify the efferocytosis rate of IDMs and MDMs allowing for measurements both in the 384- and 1536-well microplate format. Validating the applicability of the assay, inhibitors of spleen tyrosine kinase (Syk) were shown to modulate efferocytosis in IDMs and MDMs with comparable pharmacology. The miniaturized cellular assay with the upscaled provision of macrophages opens new routes to pharmaceutical drug discovery in the context of efferocytosis-modulating substances.

Stat3 role in the protective effect of FXR Agonist in parenteral nutrition-associated cholestasis.

BACKGROUND AND AIMS: Parenteral nutrition (PN) in patients with intestinal failure can lead to cholestasis (PNAC). In a PNAC mouse model, farnesoid X receptor (FXR) agonist (GW4064) treatment alleviated IL-1ß-dependent cholestatic liver injury. The objective of this study was to determine whether this hepatic protection of FXR activation is mediated through IL-6-STAT3 signaling. APPROACH AND RESULTS: Hepatic apoptotic pathways [Fas-associated protein with death domain (Fas) mRNA, caspase 8 protein, and cleaved caspase 3] and IL-6-STAT3 signaling, and expression of its downstream effectors Socs1/3 were all upregulated in the mouse PNAC model (dextran sulfate sodium enterally × 4 d followed by total PN for 14 d). Il1r-/- mice were protected from PNAC in conjunction with suppression of the FAS pathway. GW4064 treatment in the PNAC mouse increased hepatic FXR binding to the Stat3 promoter, further increased STAT3 phosphorylation and upregulated Socs1 and Socs3 mRNA, and prevented cholestasis. In HepG2 cells and primary mouse hepatocytes, IL-1ß induced IL-6 mRNA and protein, which were suppressed by GW4064. In IL-1ß or phytosterols treated HepG2 and Huh7 cells, siRNA knockdown of STAT3 significantly reduced GW4064-upregulated transcription of hepatoprotective nuclear receptor subfamily 0, group B, member 2 (NR0B2) and ABCG8. CONCLUSIONS: STAT3 signaling mediated in part the protective effects of GW4064 in the PNAC mouse, and in HepG2 cells and hepatocytes exposed to either IL-1ß or phytosterols, 2 factors critical in PNAC pathogenesis. These data demonstrate that FXR agonists may mediate hepatoprotective effects in cholestasis by inducing STAT3 signaling.

Antifibrotic Drug Nintedanib Inhibits CSF1R to Promote IL-4-associated Tissue Repair Macrophages.

Profibrotic and prohomeostatic macrophage phenotypes remain ill-defined, both in vivo and in vitro, impeding the successful development of drugs that reprogram macrophages as an attractive therapeutic approach to manage fibrotic disease. The goal of this study was to reveal profibrotic and prohomeostatic macrophage phenotypes that could guide the design of new therapeutic approaches targeting macrophages to treat fibrotic disease. This study used nintedanib, a broad kinase inhibitor approved for idiopathic pulmonary fibrosis, to dissect lung macrophage phenotypes during fibrosis-linked inflammation by combining in vivo and in vitro bulk and single-cell RNA-sequencing approaches. In the bleomycin model, nintedanib drove the expression of IL-4/IL-13-associated genes important for tissue regeneration and repair at early and late time points in lung macrophages. These findings were replicated in vitro in mouse primary bone marrow-derived macrophages exposed to IL-4/IL-13 and nintedanib. In addition, nintedanib promoted the expression of IL-4/IL-13 pathway genes in human macrophages in vitro. The molecular mechanism was connected to inhibition of the colony stimulating factor 1 (CSF1) receptor in both human and mouse macrophages. Moreover, nintedanib counterbalanced the effects of TNF on IL-4/IL-13 in macrophages to promote expression of IL-4/IL-13-regulated tissue repair genes in fibrotic contexts in vivo and in vitro. This study demonstrates that one of nintedanib's antifibrotic mechanisms is to increase IL-4 signaling in macrophages through inhibition of the CSF1 receptor, resulting in the promotion of tissue repair phenotypes.

Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis.

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct contact between macrophages and senescent cells. SCES involved augmented senescent cell expression of CD47 coinciding with increased CD47-modifying enzymes QPCT/L. SCES was reversible by interfering with the SIRPα-CD47-SHP-1 axis or QPCT/L activity. While CD47 expression increased in human and mouse senescent cells in vitro and in vivo, another ITIM-containing protein, CD24, contributed to SCES specifically in human epithelial senescent cells where it compensated for genetic deficiency in CD47. Thus, CD47 and CD24 link the pathogenic effects of senescent cells to homeostatic macrophage functions, such as efferocytosis, which we hypothesize must occur efficiently to maintain tissue homeostasis.

IL-36 receptor agonist and antagonist imbalance drives neutrophilic inflammation in COPD.

Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared with control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and is further induced by a viral mimetic, whereas IL-36Ra is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ, thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, which was inhibited by exogenous IL-36Ra. The use of a therapeutic antibody that inhibits binding to the IL-36R attenuated IL-36γ-driven inflammation and cellular crosstalk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and have shown that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising therapeutic strategy in the treatment of COPD.

CD206+ tumor-associated macrophages cross-present tumor antigen and drive antitumor immunity.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag-directed cytotoxic CD8+ T cell responses is crucial for antitumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self-Ag and non-self-Ag, including tumor-associated Ag (TAA), as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable with cross-presenting CLEC9A+ DCs (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

Gene-selective transcription promotes the inhibition of tissue reparative macrophages by TNF.

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF's anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

Automation enables high-throughput and reproducible single-cell transcriptomics library preparation.

Next-generation sequencing (NGS) has revolutionized genomics, decreasing sequencing costs and allowing researchers to draw correlations between diseases and DNA or RNA changes. Technical advances have enabled the analysis of RNA expression changes between single cells within a heterogeneous population, known as single-cell RNA-seq (scRNA-seq). Despite resolving transcriptomes of cellular subpopulations, scRNA-seq has not replaced RNA-seq, due to higher costs and longer hands-on time. Here, we developed an automated workflow to increase throughput (up to 48 reactions) and to reduce by 75% the hands-on time of scRNA-seq library preparation, using the 10X Genomics Single Cell 3' kit. After gel bead-in-emulsion (GEM) generation on the 10X Genomics Chromium Controller, cDNA amplification was performed, and the product was normalized and subjected to either the manual, standard library preparation method or a fully automated, walk-away method using a Biomek i7 Hybrid liquid handler. Control metrics showed that both quantity and quality of the single-cell gene expression libraries generated were equivalent in size and yield. Key scRNA-seq downstream quality metrics, such as unique molecular identifiers count, mitochondrial RNA content, and cell and gene counts, further showed high correlations between automated and manual workflows. Using the UMAP dimensionality reduction technique to visualize all cells, we were able to further correlate the results observed between the manual and automated methods (R=0.971). The method developed here allows for the fast, error-free, and reproducible multiplex generation of high-quality single-cell gene expression libraries.

Interrupting tumor necrosis factor-alpha signaling prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND: We have recently reported a mouse model of PN-associated cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid, bilirubin and sterol transporters Abcb11, Abcc2 and Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice. METHODS: First, recombinant TNFα was administered to mice as well as in hepatocyte cell culture. Second, Tnfr1/2KO or wild-type (WT) mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN). Finally, WT/DSS-PN mice were also infused with infliximab at 10 mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and bilirubin. RESULTS: Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed messenger RNA (mRNA) transcription of bile (Abcb11, Abcc2]) and sterol transporters (Abcg5/8) and their regulators Nr1h3 and Nr1h4. DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3, and Nr1h4. In contrast, PNAC development was prevented and mRNA expression normalized in both Tnfr1/2KO /DSS-PN mice and DSS-PN mice treated with infliximab. CONCLUSIONS: TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2, and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation.

Pharmacologic activation of hepatic farnesoid X receptor prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1ß derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model. APPROACH AND RESULTS: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1ß exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model. CONCLUSIONS: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

NF-κB Regulation of LRH-1 and ABCG5/8 Potentiates Phytosterol Role in the Pathogenesis of Parenteral Nutrition-Associated Cholestasis.

BACKGROUND AND AIMS: Chronically administered parenteral nutrition (PN) in patients with intestinal failure carries the risk for developing PN-associated cholestasis (PNAC). We have demonstrated that farnesoid X receptor (FXR) and liver X receptor (LXR), proinflammatory interleukin-1 beta (IL-1ß), and infused phytosterols are important in murine PNAC pathogenesis. In this study we examined the role of nuclear receptor liver receptor homolog 1 (LRH-1) and phytosterols in PNAC. APPROACH AND RESULTS: In a C57BL/6 PNAC mouse model (dextran sulfate sodium [DSS] pretreatment followed by 14 days of PN; DSS-PN), hepatic nuclear receptor subfamily 5, group A, member 2/LRH-1 mRNA, LRH-1 protein expression, and binding of LRH-1 at the Abcg5/8 and Cyp7a1 promoter was reduced. Interleukin-1 receptor-deficient mice (Il-1r-/- /DSS-PN) were protected from PNAC and had significantly increased hepatic mRNA and protein expression of LRH-1. NF-κB activation and binding to the LRH-1 promoter were increased in DSS-PN PNAC mice and normalized in Il-1r-/- /DSS-PN mice. Knockdown of NF-κB in IL-1ß-exposed HepG2 cells increased expression of LRH-1 and ABCG5. Treatment of HepG2 cells and primary mouse hepatocytes with an LRH-1 inverse agonist, ML179, significantly reduced mRNA expression of FXR targets ATP binding cassette subfamily C member 2/multidrug resistance associated protein 2 (ABCC2/MRP2), nuclear receptor subfamily 0, groupB, member 2/small heterodimer partner (NR0B2/SHP), and ATP binding cassette subfamily B member 11/bile salt export pump (ABCB11/BSEP). Co-incubation with phytosterols further reduced expression of these genes. Similar results were obtained by suppressing the LRH-1 targets ABCG5/8 by treatment with small interfering RNA, IL-1ß, or LXR antagonist GSK2033. Liquid chromatography-mass spectrometry and chromatin immunoprecipitation experiments in HepG2 cells showed that ATP binding cassette subfamily G member 5/8 (ABCG5/8) suppression by GSK2033 increased the accumulation of phytosterols and reduced binding of FXR to the SHP promoter. Finally, treatment with LRH-1 agonist, dilauroyl phosphatidylcholine (DLPC) protected DSS-PN mice from PNAC. CONCLUSIONS: This study suggests that NF-κB regulation of LRH-1 and downstream genes may affect phytosterol-mediated antagonism of FXR signaling in the pathogenesis of PNAC. LRH-1 could be a potential therapeutic target for PNAC.

Lactate and IL6 define separable paths of inflammatory metabolic adaptation.

Lactate is an end point of Warburg-type metabolism found in inflammatory macrophages. Recently, lactate was shown to modify histones of lipopolysaccharide (LPS)-activated macrophages in a time-dependent way and promote the expression of genes linked to tissue repair, including arginase-1 (Arg1). We tested the interrelationships between histone lactylation (Kla) and tissue reparative gene expression and found that Kla was uncoupled from changes in gene expression linked to resolving M2 macrophage activation but correlated with Arg1 expression. LPS-induced Arg1 was instead dependent on autocrine-paracrine interleukin-6 (IL6) production, the IL6 receptor, and Stat3 signal transduction. We found that Kla increases as macrophages prepare to die under inflammatory stress, and Kla was absent in macrophages that cannot generate reactive nitrogen or have defects in diverse macrophage death pathways. Thus, Kla is a consequence rather than a cause of macrophage activation but occurs coincidently with an IL6- and Arg1-dependent metabolic rewiring under inflammatory duress.

Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice.

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.

Hepatic thermal injury promotes colorectal cancer engraftment in C57/black 6 mice.

Treatment options for liver metastases (primarily colorectal cancer) are limited by high recurrence rates and persistent tumor progression. Surgical approaches to management of these metastases typically use heat energy including electrocautery, argon beam coagulation, thermal ablation of surgical margins for hemostasis, and preemptive thermal ablation to prevent bleeding or to effect tumor destruction. Based on high rates of local recurrence, these studies assess whether local effects of hepatic thermal injury (HTI) might contribute to poor outcomes by promoting a hepatic microenvironment favorable for tumor engraftment or progression due to induction of procancer cytokines and deleterious immune infiltrates at the site of thermal injury. To test this hypothesis, an immunocompetent mouse model was developed wherein HTI was combined with concomitant intrasplenic injection of cells from a well-characterized MC38 colon carcinoma cell line. In this model, HTI resulted in a significant increase in engraftment and progression of MC38 tumors at the site of thermal injury. Furthermore, there were local increases in expression of messenger ribonucleic acid (mRNA) for hypoxia-inducible factor-1α (HIF1α), arginase-1, and vascular endothelial growth factor α and activation changes in recruited macrophages at the HTI site but not in untreated liver tissue. Inhibition of HIF1α following HTI significantly reduced discreet hepatic tumor development (P = 0.03). Taken together, these findings demonstrate that HTI creates a favorable local environment that is associated with protumorigenic activation of macrophages and implantation of circulating tumors. Discrete targeting of HIF1α signaling or inhibiting macrophages offers potential strategies for improving the outcome of surgical management of hepatic metastases where HTI is used.

Author Correction: The gut microbiota in infants of obese mothers increases inflammation and susceptibility to NAFLD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunotolerant p50/NFκB Signaling and Attenuated Hepatic IFNß Expression Increases Neonatal Sensitivity to Endotoxemia.

Sepsis is a major cause of neonatal morbidity and mortality. The current paradigm suggests that neonatal susceptibility to infection is explained by an innate immune response that is functionally immature. Recent studies in adults have questioned a therapeutic role for IFNß in sepsis; however, the role of IFNß in mediating neonatal sensitivity to sepsis is unknown. We evaluated the transcriptional regulation and expression of IFNß in early neonatal (P0) and adult murine models of endotoxemia (IP LPS, 5 mg/kg). We found that hepatic, pulmonary, and serum IFNß expression was significantly attenuated in endotoxemic neonates when compared to similarly exposed adults. Furthermore, endotoxemia induced hepatic p65/NFκB and IRF3 activation exclusively in adults. In contrast, endotoxemia induced immunotolerant p50/NFκB signaling in neonatal mice without evidence of IRF3 activation. Consistent with impaired IFNß expression and attenuated circulating serum levels, neonatal pulmonary STAT1 signaling and target gene expression was significantly lower than adult levels. Using multiple in vivo approaches, the source of hepatic IFNß expression in endotoxemic adult mice was determined to be the hepatic macrophage, and experiments in RAW 264.7 cells confirmed that LPS-induced IFNß expression was NFκB dependent. Finally, treating neonatal mice with IFNß 2 h after endotoxemia stimulated pulmonary STAT1 signaling and STAT1 dependent gene expression. Furthermore, IFNß treatment of endotoxemic neonatal animals resulted in significantly improved survival following exposure to lethal endotoxemia. In conclusion, endotoxemia induced IFNß expression is attenuated in the early neonatal period, secondary to impaired NFκB-p65/IRF3 signaling. Pre-treatment with IFNß decreases neonatal sensitivity to endotoxemia. These results support further study of the role of impaired IFNß expression and neonatal sensitivity to sepsis.