mTORC1 Signaling Regulates Proinflammatory Macrophage Function and Metabolism.

Metabolic programming is integrally linked to immune cell function. Nowhere is this clearer than in the differentiation of macrophages. Proinflammatory M1 macrophages primarily use glycolysis as a rapid energy source but also to generate antimicrobial compounds, whereas alternatively activated M2 macrophages primarily rely on oxidative phosphorylation for the longevity required for proper wound healing. mTOR signaling has been demonstrated to be a key regulator of immune cell metabolism and function. mTORC2 signaling is required for the generation of M2 macrophages, whereas the role of mTORC1 signaling, a key regulator of glycolysis, has been controversial. By using genetic deletion of mTORC1 signaling in C57BL/6 mouse macrophages, we observed enhanced M1 macrophage function in vitro and in vivo. Surprisingly, this enhancement occurred despite a significant defect in M1 macrophage glycolytic metabolism. Mechanistically, enhanced M1 function occurred because of inhibition of the class III histone deacetylases the sirtuins, resulting in enhanced histone acetylation. Our findings provide a counterpoint to the paradigm that enhanced immune cell function must occur in the presence of increased cellular metabolism and identifies a potential, pharmacologic target for the regulation of inflammatory responses.

An Fc domain protein-small molecule conjugate as an enhanced immunomodulator.

Proteins as well as small molecules have demonstrated success as therapeutic agents, but their pharmacologic properties sometimes fall short against particular drug targets. Although the adenosine 2a receptor (A(2A)R) has been identified as a promising target for immunotherapy, small molecule A(2A)R agonists have suffered from short pharmacokinetic half-lives and the potential for toxicity by modulating nonimmune pathways. To overcome these limitations, we have tethered the A(2A)R agonist CGS-21680 to the immunoglobulin Fc domain using expressed protein ligation with Sf9 cell secreted protein. The protein small molecule conjugate Fc-CGS retained potent Fc receptor and A(2A)R interactions and showed superior properties as a therapeutic for the treatment of a mouse model of autoimmune pneumonitis. This approach may provide a general strategy for optimizing small molecule therapeutics.

H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal K(ATP) channel.

Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)-the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1-10mM). GYY4137 slowly released appreciable levels of H2S in the range of 10-275 µM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

Pulmonary delivery of docosahexaenoic acid mitigates bleomycin-induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis is an untreatable, fatal disease characterized by excess deposition of extracellular matrix and inflammation. Although the etiology of pulmonary fibrosis is unknown, recent studies have implicated dysregulated immune responses and wound healing. Since n-3 polyunsaturated fatty acids (n-3 PUFAs) may beneficially modulate immune responses in a variety of inflammatory disorders, we investigated the therapeutic role of docosahexaenoic acid (DHA), a single n-3 PUFA, in lung fibrosis. METHODS: Intratracheal DHA or PBS was administered to mouse lungs 4 days prior to intratracheal bleomycin treatment. Body weight and survival were monitored for 21 days. Bronchoalveolar fluid (BALF) and lung inflammatory cells, cytokines, eicosanoids, histology and lung function were determined on serial days (0, 3, 7, 14, 21) after bleomycin injury. RESULTS: Intratracheal administration of DHA mitigated bleomycin-induced lung injury. Mice pretreated with DHA had significantly less weight loss and mortality after bleomycin injury. The lungs from DHA-pretreated mice had markedly less fibrosis. DHA pretreatment also protected the mice from the functional changes associated with bleomycin injury. Bleomycin-induced cellular inflammation in BALF and lung tissue was blunted by DHA pretreatment. These advantageous effects of DHA pretreatment were associated with decreased IL-6, LTB4, PGE2 and increased IL-10. CONCLUSIONS: Our findings demonstrate that intratracheal administration of DHA, a single PUFA, protected mice from the development of bleomycin-induced pulmonary inflammation and fibrosis. These results suggest that further investigations regarding the role of n-3 polyunsaturated fatty acids in fibrotic lung injury and repair are needed.

Murine Model of Airway Fibrosis has Anatomic, Physiologic, and Molecular Congruency to Human iSGS.

OBJECTIVE: To narrow knowledge gaps in the pathophysiology of idiopathic subglottic stenosis (iSGS) through comparison of a murine subglottic stenosis model with iSGS. STUDY DESIGN: In vivo animal study. SETTING: Academic institution. METHODS: Murine samples/measurements were obtained from mice that underwent chemomechanical injury with a wire brush and bleomycin. Human samples/measurements were obtained from iSGS patients. Anatomic, physiologic, and epithelial molecular data were collected using histology, human peak expiratory flow (PEF) and murine airway conductance, gene expression analysis with quantitative polymerase chain reaction, and protein analysis with quantitative immunohistochemistry. RESULTS: Anatomic patterns of scars at the subglottis and proximal trachea seen in the murine model are similar to iSGS patients. Subglottic stenosis (SGS) mice had a decrease (P = .0194) in airway conductance compared to healthy controls, similar to a decrease (P = .0001) in predilation PEF versus postdilation in iSGS patients. There was decreased epithelial gene expression of E-cadherin (ECAD) (P < 0.01), occludin (OCLN) (P < .01), and cytokeratin-5 (CK5) (P < .05) and protein expression of ECAD (H/M: P < .001), OCLN (H: P < 0.05, M: P < .001), and CK5 (H: P < .001, M: P < .01) in murine SGS and iSGS versus controls. CONCLUSION: The murine SGS model shows anatomic, physiologic, and molecular congruency with human iSGS, making it a reasonable model to investigate iSGS. The molecular similarities in epithelial barrier dysfunction suggest it may best be suited to explore epithelial mechanisms of iSGS and therapies directed at epithelial reconstitution. This model provides a foundation to collect data that will improve understanding of iSGS, and, ultimately, translate into more accurate animal models for future use.

Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis.

OBJECTIVES: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. METHODS: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). RESULTS: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. CONCLUSION: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. LEVEL OF EVIDENCE: NA Laryngoscope, 134:374-381, 2024.

A Comprehensive Flow Cytometry Panel for Analysis of Idiopathic Subglottic Stenosis.

OBJECTIVE: To present a comprehensive flow cytometry panel for idiopathic subglottic stenosis (iSGS). STUDY DESIGN: Controlled ex vivo cohort study. SETTING: Tertiary care academic hospital in a metropolitan area. METHODS: Flow cytometry and single-cell RNA sequencing were performed on 9 paired normal and scar tissue samples from iSGS patients. Flow cytometry was used to assess the presence of myeloid (CD11b, CD14, CD15, Siglec8), lymphoid (CD3, CD4, CD8, gamma delta [γδ], FOXP3), endothelial (CD31), fibroblast (CD90, SMA), and epithelial (CD326, CK5) markers. RESULTS: On flow cytometry, iSGS scar is characterized by an increased presence of myeloid, lymphoid, endothelial, and fibroblast cell types, but a decreased presence of epithelial cells. In the myeloid lineage, iSGS scar samples demonstrated increased CD11b+ monocytes (P < .001), Siglec8+ eosinophils (P = .03), and CD14+ monocytes (P = .02). In the lymphoid lineage, iSGS scar demonstrated increased CD3+ T-cells (P < .001), CD4+ helper T-cells (P < .001), γδ+ T-cells (P < .001), and FOXP3+ regulatory T-cells (P = .002). iSGS scar exhibited specific increases in CD90+ (P = .04) and SMA+ (P < .001) fibroblasts but decreased CD326+ (E-cadherin) epithelial cells (P = .01) relative to normal samples. CONCLUSION: We present a comprehensive flow cytometry panel for iSGS. This flow panel may serve as a common platform among airway scientists to elucidate the cellular mechanisms underpinning iSGS and other upper airway pathologies. Scar iSGS samples demonstrate a distinct cellular profile relative to normal iSGS specimens, exhibiting increased fibroblast, endothelial, and inflammatory cell types but decreased epithelium.

An Ovine Model Yields Histology and Gene Expression Changes Consistent with Laryngotracheal Stenosis.

OBJECTIVES: Animal models for laryngotracheal stenosis (LTS) are critical to understand underlying mechanisms and study new therapies. Current animal models for LTS are limited by small airway sizes compared to human. The objective of this study was to develop and validate a novel, large animal ovine model for LTS. METHODS: Sheep underwent either bleomycin-coated polypropylene brush injury to the subglottis (n = 6) or airway stent placement (n = 2) via suspension microlaryngoscopy. Laryngotracheal complexes were harvested 4 weeks following injury or stent placement. For the airway injury group, biopsies (n = 3 at each site) were collected of tracheal scar and distal normal regions, and analyzed for fibrotic gene expression. Lamina propria (LP) thickness was compared between injured and normal areas of trachea. RESULTS: No mortality occurred in sheep undergoing airway injury or stent placement. There was no migration of tracheal stents. After protocol optimization, LP thickness was significantly increased in injured trachea (Sheep #3: 529.0 vs. 850.8 um; Sheep #4: 933.0 vs. 1693.2 um; Sheep #5: 743.7 vs. 1378.4 um; Sheep #6: 305.7 vs. 2257.6 um). A significant 62-fold, 20-fold, 16-fold, 16-fold, and 9-fold change of COL1, COL3, COL5, FN1, and TGFB1 was observed in injured scar specimen relative to unaffected airway, respectively. CONCLUSION: An ovine LTS model produces histologic and transcriptional changes consistent with fibrosis seen in human LTS. Airway stent placement in this model is safe and feasible. This large airway model is a reliable and reproducible method to assess the efficacy of novel LTS therapies prior to clinical translation. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

Adoptive transfer of CD49a+ Tissue resident memory cells reverses pulmonary fibrosis in mice.

Pulmonary fibrosis is a devastating disease with no effective treatments to cure, stop or reverse the unremitting, fatal fibrosis. A critical barrier to treating this disease is the lack of understanding of the pathways leading to fibrosis as well as those regulating the resolution of fibrosis. Fibrosis is the pathologic side of normal tissue repair that results when the normal wound healing programs go awry. Successful resolution of tissue injury requires several highly coordinated pathways, and this research focuses on the interplay between these overlapping pathways: immune effectors, inflammatory mediators and fibroproliferation in the resolution of fibrosis. Previously we have successfully prevented, mitigated, and even reversed established fibrosis using vaccinia vaccination immunotherapy in two models of murine lung fibrosis. The mechanism by which vaccinia reverses fibrosis is by vaccine induced lung specific Th1 skewed tissue resident memory (TRMs) in the lung. In this study, we isolated a population of vaccine induced TRMs - CD49a+ CD4+ T cells - that are both necessary and sufficient to reverse established pulmonary fibrosis. Using adoptive cellular therapy, we demonstrate that intratracheal administration of CD49a+ CD4+ TRMs into established fibrosis, reverses the fibrosis histologically, by promoting a decrease in collagen, and functionally, by improving lung function, without the need for vaccination. Furthermore, co-culture of in vitro derived CD49+ CD4+ human TRMs with human fibroblasts from individuals with idiopathic pulmonary fibrosis (IPF) results in the down regulation of IPF fibroblast collagen production. Lastly, we demonstrate in human IPF lung histologic samples that CD49a+ CD4+ TRMs, which can down regulate human IPF fibroblast function, fail to increase in the IPF lungs, thus potentially failing to promote resolution. Thus, we define a novel unappreciated role for tissue resident memory T cells in regulating established lung fibrosis to promote resolution of fibrosis and re-establish lung homeostasis. We demonstrate that immunotherapy, in the form of adoptive transfer of CD49a+ CD4+ TRMs into the lungs of mice with established fibrosis, not only stops progression of the fibrosis but more importantly reverses the fibrosis. These studies provide the insight and preclinical rationale for a novel paradigm shifting approach of using cellular immunotherapy to treat lung fibrosis.

Myeloid Phenotypes in Tracheostomy-Associated Granulation Tissue.

OBJECTIVE(S): Tracheostomy-associated granulation tissue is a common, recurrent problem occurring secondary to chronic mucosal irritation. Although granulation tissue is composed of predominantly innate immune cells, the phenotype of monocytes and macrophages in tracheostomy-associated granulation tissue is unknown. This study aims to define the myeloid cell population in granulation tissue secondary to tracheostomy. METHODS: Granulation tissue biopsies were obtained from 8 patients with tracheostomy secondary to laryngotracheal stenosis. Cell type analysis was performed by flow cytometry and gene expression was measured by quantitative real-time polymerase chain reaction. These methods and immunohistochemistry were used to define the monocyte/macrophage population in granulation tissue and were compared to tracheal autopsy control specimens. RESULTS: Flow cytometry demonstrated macrophages (CD45+CD11b+) and monocytes (CD45+FSClow SSClow ) represent 23.2 ± 6% of the granulation tissue cell population. The M2 phenotype (CD206) is present in 77 ± 11% of the macrophage population and increased compared to the M1 phenotype (p = 0.012). Classical monocytes (CD45+CD14high CD16low ) were increased in granulation tissue compared to controls (61.2 ± 7% and 30 ± 8.5%, p = 0.038). Eighty-five percent of macrophages expressed pro-inflammatory S100A8/A9 and 36 ± 4% of macrophages co-localized CD169, associated with tissue-resident macrophages. M2 gene expression (Arg1/CD206) was increased in granulation tissue (3.7 ± 0.4, p = 0.035 and 3.5 ± 0.5, p = 0.047) whereas M1 gene expression (CD80/CD86) was similar to controls (p = 0.64, p = 0.3). Immunohistochemistry of granulation tissue demonstrated increased cells co-localizing CD11b and CD206. CONCLUSIONS: M2 macrophages are the dominant macrophage phenotype in tracheostomy-associated granulation tissue. The role of this cell type in promoting ongoing inflammation warrants future investigation to identify potential treatments for granulation tissue secondary to tracheostomy. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2346-2356, 2023.

Sirolimus-eluting airway stent reduces profibrotic Th17 cells and inhibits laryngotracheal stenosis.

Laryngotracheal stenosis (LTS) is pathologic fibrotic narrowing of the larynx and trachea characterized by hypermetabolic fibroblasts and CD4+ T cell-mediated inflammation. However, the role of CD4+ T cells in promoting LTS fibrosis is unknown. The mTOR signaling pathways have been shown to regulate the T cell phenotype. Here we investigated the influence of mTOR signaling in CD4+ T cells on LTS pathogenesis. In this study, human LTS specimens revealed a higher population of CD4+ T cells expressing the activated isoform of mTOR. In a murine LTS model, targeting mTOR with systemic sirolimus and a sirolimus-eluting airway stent reduced fibrosis and Th17 cells. Selective deletion of mTOR in CD4+ cells reduced Th17 cells and attenuated fibrosis, demonstrating CD4+ T cells' pathologic role in LTS. Multispectral immunofluorescence of human LTS revealed increased Th17 cells. In vitro, Th17 cells increased collagen-1 production by LTS fibroblasts, which was prevented with sirolimus pretreatment of Th17 cells. Collectively, mTOR signaling drove pathologic CD4+ T cell phenotypes in LTS, and targeting mTOR with sirolimus was effective at treating LTS through inhibition of profibrotic Th17 cells. Finally, sirolimus may be delivered locally with a drug-eluting stent, transforming clinical therapy for LTS.

Serendipitous Discovery of T Cell-Produced KLK1b22 as a Regulator of Systemic Metabolism.

In order to study mechanistic/mammalian target of rapamycin's role in T cell differentiation, we generated mice in which Rheb is selectively deleted in T cells (T-Rheb-/- C57BL/6J background). During these studies, we noted that T-Rheb-/- mice were consistently heavier but had improved glucose tolerance and insulin sensitivity as well as a marked increase in beige fat. Microarray analysis of Rheb-/- T cells revealed a marked increase in expression of kallikrein 1-related peptidase b22 (Klk1b22). Overexpression of KLK1b22 in vitro enhanced insulin receptor signaling, and systemic overexpression of KLK1b22 in C57BL/6J mice also enhances glucose tolerance. Although KLK1B22 expression was markedly elevated in the T-Rheb-/- T cells, we never observed any expression in wild-type T cells. Interestingly, in querying the mouse Immunologic Genome Project, we found that Klk1b22 expression was also increased in wild-type 129S1/SVLMJ and C3HEJ mice. Indeed, both strains of mice demonstrate exceptionally improved glucose tolerance. This prompted us to employ CRISPR-mediated knockout of KLK1b22 in 129S1/SVLMJ mice, which in fact led to reduced glucose tolerance. Overall, our studies reveal (to our knowledge) a novel role for KLK1b22 in regulating systemic metabolism and demonstrate the ability of T cell-derived KLK1b22 to regulate systemic metabolism. Notably, however, further studies have revealed that this is a serendipitous finding unrelated to Rheb.

Hyaluronan fragments promote inflammation by down-regulating the anti-inflammatory A2a receptor.

The tissue microenvironment plays a critical role in regulating inflammation. Chronic inflammation leads to an influx of inflammatory cells and mediators, extracellular matrix turnover, and increased extracellular adenosine. Low molecular weight (LMW) fragments of hyaluronan (HA), a matrix component, play a critical role in lung inflammation and fibrosis by inducing inflammatory gene expression at the injury site. Adenosine, a crucial negative regulator of inflammation, protects tissues from immune destruction via the adenosine A2a receptor (A2aR). Therefore, these two extracellular products of inflammation play opposing roles in regulating immune responses. As such, we wanted to determine the effect of LMW HA on A2aR function. In this article, we demonstrate that LMW HA causes a rapid, significant, and sustained down-regulation of the A2aR. CD44 was found to be necessary for LMW HA to down-modulate the A2aR as was protein kinase C signaling. We also demonstrate that LMW HA induces A2aR down-regulation during inflammation in vivo, and that this down-regulation can be blocked by treatment with an HA-blocking peptide. Because adenosine plays a critical role in limiting inflammation, our data provide a novel mechanism whereby LMW HA itself may further augment inflammation. By defining the pro- and anti-inflammatory properties of extracellular matrix components, we will be better able to identify specific pharmacologic targets as potential therapies.

A unique proteolytic fragment of alpha1-antitrypsin is elevated in ductal fluid of breast cancer patient.

By comparison of mass spectra from a small cohort of nipple aspiration fluids (NAF), we previously discovered a panel of five candidate breast cancer biomarkers among them an unidentified 4.7 kD peptide BF5. The purposes of the present study were to verify the presence of BF5 in an independent cohort; to determine the protein identity of BF5; and to provide insight into the biology of BF5 production and elevation in tumor-associated NAF. We prospectively collected bilaterally matched NAF from patients with unilateral Stage I/II breast cancer (IBC-31), ductal carcinoma in situ (DCIS-6), atypical ductal hyperplasia (ADH-5), and presumed healthy women who came to routine mammography and had a normal exam (31). Following the consolidation of its cancer-associated expression on SELDI-mass spectrometry, BF5 was isolated by gel electrophoresis and sequenced by tandem mass spectrometry. BF5 was elevated in 15-25% of women with IBC, DCIS, or ADH vs. 0% of controls. This elevation was restricted to the affected breasts. BF5 was identified as 41/42-aa C-terminal peptide of alpha1-antitrypsin (AAT), the principle inhibitor of serine protease neutrophile elastase. The full length AAT showed a consistent expression pattern as C-41/42, and C-41/42 can be generated in vitro by MMP-7 cleavage. In conclusion, elevated C-41/42 is likely the result of elevated AAT synthesis, and the activity of specific MMPs present within the tumor. As other C-terminal fragments of AAT are reported to function as tumor-derived suppressors to the host immune-system, elevated C-41/42 may also be predictive of a poor outcome.

Targeting glutamine metabolism enhances tumor-specific immunity by modulating suppressive myeloid cells.

Myeloid cells comprise a major component of the tumor microenvironment (TME) that promotes tumor growth and immune evasion. By employing a small-molecule inhibitor of glutamine metabolism, not only were we able to inhibit tumor growth, but we markedly inhibited the generation and recruitment of myeloid-derived suppressor cells (MDSCs). Targeting tumor glutamine metabolism led to a decrease in CSF3 and hence recruitment of MDSCs as well as immunogenic cell death, leading to an increase in inflammatory tumor-associated macrophages (TAMs). Alternatively, inhibiting glutamine metabolism of the MDSCs themselves led to activation-induced cell death and conversion of MDSCs to inflammatory macrophages. Surprisingly, blocking glutamine metabolism also inhibited IDO expression of both the tumor and myeloid-derived cells, leading to a marked decrease in kynurenine levels. This in turn inhibited the development of metastasis and further enhanced antitumor immunity. Indeed, targeting glutamine metabolism rendered checkpoint blockade-resistant tumors susceptible to immunotherapy. Overall, our studies define an intimate interplay between the unique metabolism of tumors and the metabolism of suppressive immune cells.

The adenosine a2a receptor inhibits matrix-induced inflammation in a novel fashion.

Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.

Inhibition of glutamine metabolism accelerates resolution of acute lung injury.

Despite recent advances, acute respiratory distress syndrome (ARDS) remains a severe and often fatal disease for which there is no therapy able to reduce the underlying excessive lung inflammation or enhance resolution of injury. Metabolic programming plays a critical role in regulating inflammatory responses. Due to their high metabolic needs, neutrophils, macrophages, and lymphocytes rely upon glutamine metabolism to support activation and function. Additionally, during times of physiologic stress, nearly all cells, including fibroblasts and epithelial cells, require glutamine metabolism. We hypothesized that inhibiting glutamine metabolism reduces lung inflammation and promotes resolution of acute lung injury. Lung injury was induced by instilling lipopolysaccharide (LPS) intratracheally. To inhibit glutamine metabolism, we administered a glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON) that binds to glutamine-utilizing enzymes and transporters, after injury was well established. Treatment with DON led to less lung injury, fewer lung neutrophils, lung inflammatory and interstitial macrophages, and lower levels of proinflammatory cytokines and chemokines at 5 and/or 7 days after injury. Additionally, DON led to earlier expression of the growth factor amphiregulin and more rapid recovery of LPS-induced weight loss. Thus, DON reduced lung inflammation and promoted resolution of injury. These data contribute to our understanding of how glutamine metabolism regulates lung inflammation and repair, and identifies a novel target for future therapies for ARDS and other inflammatory lung diseases.

mTORC2 Signaling Selectively Regulates the Generation and Function of Tissue-Resident Peritoneal Macrophages.

Tissue-resident macrophages play critical roles in sentinel and homeostatic functions as well as in promoting inflammation and immunity. It has become clear that the generation of these cells is highly dependent upon tissue-specific cues derived from the microenvironment that, in turn, regulate unique differentiation programs. Recently, a role for GATA6 has emerged in the differentiation programming of resident peritoneal macrophages. We identify a critical role for mTOR in integrating cues from the tissue microenvironment in regulating differentiation and metabolic reprogramming. Specifically, inhibition of mTORC2 leads to enhanced GATA6 expression in a FOXO1 dependent fashion. Functionally, inhibition of mTORC2 promotes peritoneal resident macrophage generation in the resolution phase during zymosan-induced peritonitis. Also, mTORC2-deficient peritoneal resident macrophages displayed increased functionality and metabolic reprogramming. Notably, mTORC2 activation distinguishes tissue-resident macrophage proliferation and differentiation from that of M2 macrophages. Overall, our data implicate a selective role for mTORC2 in the differentiation of tissue-resident macrophages.

CNS-targeted autoimmunity leads to increased influenza mortality in mice.

The discovery that central nervous system (CNS)-targeted autoreactive T cells required a process of licensing in the lung revealed an unexpected relationship between these organs. The clinical and immunological significance of this finding is bidirectional in that it showed not only a mechanism by which T cells become pathogenic before entering the CNS, but also the potential for this process to influence lung immunity as well. Epidemiological studies have shown that people with multiple sclerosis (MS) suffer from increased morbidity and mortality from infectious diseases, independent of immunosuppressive therapies. Respiratory infections account for a large percentage of deaths of people with MS. In this study, to investigate the mechanisms responsible for this enhanced susceptibility, we established a comorbid model system in which mice with experimental autoimmune encephalomyelitis (EAE) were administered a sublethal dose of influenza. Whereas mice with either EAE alone or influenza alone survived, 70% of comorbid mice died as a result of uncontrolled viral replication. Immunological analyses revealed that the induction of EAE led to a surprising alteration of the lung milieu, converting an effective stimulatory influenza-reactive environment into a suppressive one. These results provide mechanistic information that may help to explain the unexpected immunological interactions.

Vaccinia vaccine-based immunotherapy arrests and reverses established pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease without any cure. Both human disease and animal models demonstrate dysregulated wound healing and unregulated fibrogenesis in a background of low-grade chronic T lymphocyte infiltration. Tissue-resident memory T cells (Trm) are emerging as important regulators of the immune microenvironment in response to pathogens, and we hypothesized that they might play a role in regulating the unremitting inflammation that promotes lung fibrosis. Herein, we demonstrate that lung-directed immunotherapy, in the form of i.n. vaccination, induces an antifibrotic T cell response capable of arresting and reversing lung fibrosis. In mice with established lung fibrosis, lung-specific T cell responses were able to reverse established pathology - as measured by decreased lung collagen, fibrocytes, and histologic injury - and improve physiologic function. Mechanistically, we demonstrate that this effect is mediated by vaccine-induced lung Trm. These data not only have implications for the development of immunotherapeutic regimens to treat IPF, but also suggest a role for targeting tissue-resident memory T cells to treat other tissue-specific inflammatory/autoimmune disorders.