Augmentation of Endothelial S1PR1 Attenuates Postviral Pulmonary Fibrosis.

Respiratory viral infections are frequent causes of acute respiratory distress syndrome (ARDS), a disabling condition with a mortality of up to 46%. The pulmonary endothelium plays an important role in the development of ARDS as well as the pathogenesis of pulmonary fibrosis; however, the therapeutic potential to modulate endothelium-dependent signaling to prevent deleterious consequences has not been well explored. Here, we used a clinically relevant influenza A virus infection model, endothelial cell-specific transgenic gain-of-function and loss-of-function mice as well as pharmacologic approaches and in vitro modeling, to define the mechanism by which S1PR1 expression is dampened during influenza virus infection and determine whether therapeutic augmentation of S1PR1 has the potential to reduce long-term postviral fibrotic complications. We found that the influenza virus-induced inflammatory milieu promoted internalization of S1PR1, which was pharmacologically inhibited with paroxetine, an inhibitor of GRK2. Moreover, genetic overexpression or administration of paroxetine days after influenza virus infection was sufficient to reduce postviral pulmonary fibrosis. Taken together, our data suggest that endothelial S1PR1 signaling provides critical protection against long-term fibrotic complications after pulmonary viral infection. These findings support the development of antifibrotic strategies that augment S1PR1 expression in virus-induced ARDS to improve long-term patient outcomes.

Regulatory T cell-derived IL-1Ra suppresses the innate response to respiratory viral infection.

Regulatory T (Treg) cell modulation of adaptive immunity and tissue homeostasis is well described; however, less is known about Treg cell-mediated regulation of the innate immune response. Here we show that deletion of ST2, the receptor for interleukin (IL)-33, on Treg cells increased granulocyte influx into the lung and increased cytokine production by innate lymphoid and γδ T cells without alteration of adaptive immunity to influenza. IL-33 induced high levels of the interleukin-1 receptor antagonist (IL-1Ra) in ST2+ Treg cells and deletion of IL-1Ra in Treg cells increased granulocyte influx into the lung. Treg cell-specific deletion of ST2 or IL-1Ra improved survival to influenza, which was dependent on IL-1. Adventitial fibroblasts in the lung expressed high levels of the IL-1 receptor and their chemokine production was suppressed by Treg cell-produced IL-1Ra. Thus, we define a new pathway where IL-33-induced IL-1Ra production by tissue Treg cells suppresses IL-1-mediated innate immune responses to respiratory viral infection.

Notch4 signaling limits regulatory T-cell-mediated tissue repair and promotes severe lung inflammation in viral infections.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections.

Lung Histopathology in Coronavirus Disease 2019 as Compared With Severe Acute Respiratory Sydrome and H1N1 Influenza: A Systematic Review.

BACKGROUND: Patients with severe coronavirus disease 2019 (COVID-19) have respiratory failure with hypoxemia and acute bilateral pulmonary infiltrates, consistent with ARDS. Respiratory failure in COVID-19 might represent a novel pathologic entity. RESEARCH QUESTION: How does the lung histopathology described in COVID-19 compare with the lung histopathology described in SARS and H1N1 influenza? STUDY DESIGN AND METHODS: We conducted a systematic review to characterize the lung histopathologic features of COVID-19 and compare them against findings of other recent viral pandemics, H1N1 influenza and SARS. We systematically searched MEDLINE and PubMed for studies published up to June 24, 2020, using search terms for COVID-19, H1N1 influenza, and SARS with keywords for pathology, biopsy, and autopsy. Using PRISMA-Individual Participant Data guidelines, our systematic review analysis included 26 articles representing 171 COVID-19 patients; 20 articles representing 287 H1N1 patients; and eight articles representing 64 SARS patients. RESULTS: In COVID-19, acute-phase diffuse alveolar damage (DAD) was reported in 88% of patients, which was similar to the proportion of cases with DAD in both H1N1 (90%) and SARS (98%). Pulmonary microthrombi were reported in 57% of COVID-19 and 58% of SARS patients, as compared with 24% of H1N1 influenza patients. INTERPRETATION: DAD, the histologic correlate of ARDS, is the predominant histopathologic pattern identified in lung pathology from patients with COVID-19, H1N1 influenza, and SARS. Microthrombi were reported more frequently in both patients with COVID-19 and SARS as compared with H1N1 influenza. Future work is needed to validate this histopathologic finding and, if confirmed, elucidate the mechanistic underpinnings and characterize any associations with clinically important outcomes.

Interleukin-33 activates regulatory T cells to suppress innate γδ T cell responses in the lung.

Foxp3+ regulatory T (Treg) cells expressing the interleukin (IL)-33 receptor ST2 mediate tissue repair in response to IL-33. Whether Treg cells also respond to the alarmin IL-33 to regulate specific aspects of the immune response is not known. Here we describe an unexpected function of ST2+ Treg cells in suppressing the innate immune response in the lung to environmental allergens without altering the adaptive immune response. Following allergen exposure, ST2+ Treg cells were activated by IL-33 to suppress IL-17-producing γδ T cells. ST2 signaling in Treg cells induced Ebi3, a component of the heterodimeric cytokine IL-35 that was required for Treg cell-mediated suppression of γδ T cells. This response resulted in fewer eosinophil-attracting chemokines and reduced eosinophil recruitment into the lung, which was beneficial to the host in reducing allergen-induced inflammation. Thus, we define a fundamental role for ST2+ Treg cells in the lung as a negative regulator of the early innate γδ T cell response to mucosal injury.

Migratory DCs activate TGF-ß to precondition naïve CD8+ T cells for tissue-resident memory fate.

Epithelial resident memory T (eTRM) cells serve as sentinels in barrier tissues to guard against previously encountered pathogens. How eTRM cells are generated has important implications for efforts to elicit their formation through vaccination or prevent it in autoimmune disease. Here, we show that during immune homeostasis, the cytokine transforming growth factor ß (TGF-ß) epigenetically conditions resting naïve CD8+ T cells and prepares them for the formation of eTRM cells in a mouse model of skin vaccination. Naïve T cell conditioning occurs in lymph nodes (LNs), but not in the spleen, through major histocompatibility complex class I-dependent interactions with peripheral tissue-derived migratory dendritic cells (DCs) and depends on DC expression of TGF-ß-activating αV integrins. Thus, the preimmune T cell repertoire is actively conditioned for a specialized memory differentiation fate through signals restricted to LNs.

Peroxidase Sensitive Amplifiable Probe for Molecular Magnetic Resonance Imaging of Pulmonary Inflammation.

An amplifiable magnetic resonance imaging (MRI) probe that combines the stability of the macrocyclic Gd-DOTAGA core with a peroxidase-reactive 5-hydroxytryptamide (5-HT) moiety is reported. The incubation of the complex under enzymatic oxidative conditions led to a 1.7-fold increase in r1 at 1.4 T that was attributed to an oligomerization of the probe upon oxidation. This probe, Gd-5-HT-DOTAGA, provided specific detection of lung inflammation by MRI in bleomycin-injured mice.

Quantitative assessment of airway remodelling and response to allergen in asthma.

BACKGROUND AND OBJECTIVE: In vivo evaluation of the microstructural differences between asthmatic and non-asthmatic airways and their functional consequences is relevant to understanding and, potentially, treating asthma. In this study, we use endobronchial optical coherence tomography to investigate how allergic airways with asthma differ from allergic non-asthmatic airways in baseline microstructure and in response to allergen challenge. METHODS: A total of 45 subjects completed the study, including 20 allergic, mildly asthmatic individuals, 22 non-asthmatic allergic controls and 3 healthy controls. A 3-cm airway segment in the right middle and right upper lobe were imaged in each subject immediately before and 24 h following segmental allergen challenge to the right middle lobe. Relationships between optical airway measurements (epithelial and mucosal thicknesses, mucosal buckling and mucus) and airway obstruction (FEV1 /FVC (forced expiratory volume in 1 s/forced vital capacity) and FEV1 % (FEV1 as a percentage of predictive value)) were investigated. RESULTS: Significant increases at baseline and in response to allergen were observed for all four of our imaging metrics in the asthmatic airways compared to the non-asthmatic airways. Epithelial thickness and mucosal buckling exhibited a significant relationship to FEV1 /FVC in the asthmatic group. CONCLUSION: Simultaneous assessments of airway microstructure, buckling and mucus revealed both structural and functional differences between the mildly asthmatic and control groups, with airway buckling seeming to be the most relevant factor. The results of this study demonstrate that a comprehensive, microstructural approach to assessing the airways may be important in future asthma studies as well as in the monitoring and treatment of asthma.

Birefringence microscopy platform for assessing airway smooth muscle structure and function in vivo.

The inability to visualize airway smooth muscle (ASM) cells in vivo is a major obstacle in understanding their role in normal physiology and diseases. At present, there is no imaging modality available to assess ASM in vivo. Confocal endomicroscopy lacks the penetration depth and field of view, and conventional optical coherence tomography (OCT) does not have sufficient contrast to differentiate ASM from surrounding tissues. We have developed a birefringence microscopy platform that leverages the micro-organization of tissue to add further dimension to traditional OCT. We have used this technology to validate ASM measurements in ex vivo swine and canine studies, visualize and characterize volumetric representations of ASM in vivo, and quantify and predict ASM contractile force as a function of optical retardation. We provide in vivo images and volumetric assessments of ASM in living humans and document structural disease variations in subjects with mild asthma. The opportunity to link inflammatory responses to ASM responses and to link ASM responses to clinical responses and outcomes could lead to an increased understanding of diseases of the airway and, ultimately, to improved patient outcomes.

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation.

Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation.

The receptor TREML4 amplifies TLR7-mediated signaling during antiviral responses and autoimmunity.

The molecules and pathways that fine-tune innate inflammatory responses mediated by Toll-like receptor 7 (TLR7) remain to be fully elucidated. Using an unbiased genome-scale screen with short hairpin RNA (shRNA), we identified the receptor TREML4 as an essential positive regulator of TLR7 signaling. Macrophages from Treml4(-/-) mice were hyporesponsive to TLR7 agonists and failed to produce type I interferons due to impaired phosphorylation of the transcription factor STAT1 by the mitogen-activated protein kinase p38 and decreased recruitment of the adaptor MyD88 to TLR7. TREML4 deficiency reduced the production of inflammatory cytokines and autoantibodies in MRL/lpr mice, which are prone to systemic lupus erythematosus (SLE), and inhibited the antiviral immune response to influenza virus. Our data identify TREML4 as a positive regulator of TLR7 signaling and provide insight into the molecular mechanisms that control antiviral immunity and the development of autoimmunity.

Chemokines and chemokine receptors: positioning cells for host defense and immunity.

Chemokines are chemotactic cytokines that control the migratory patterns and positioning of all immune cells. Although chemokines were initially appreciated as important mediators of acute inflammation, we now know that this complex system of approximately 50 endogenous chemokine ligands and 20 G protein-coupled seven-transmembrane signaling receptors is also critical for the generation of primary and secondary adaptive cellular and humoral immune responses. Recent studies demonstrate important roles for the chemokine system in the priming of naive T cells, in cell fate decisions such as effector and memory cell differentiation, and in regulatory T cell function. In this review, we focus on recent advances in understanding how the chemokine system orchestrates immune cell migration and positioning at the organismic level in homeostasis, in acute inflammation, and during the generation and regulation of adoptive primary and secondary immune responses in the lymphoid system and peripheral nonlymphoid tissue.

Targeting cells in motion: migrating toward improved therapies.

The development of clinical therapeutics that interfere with the migration of leukocytes has revolutionized the treatment of multiple sclerosis and holds great promise for the treatment of a wide range of inflammatory diseases. As the molecules essential for the multi-step adhesion cascade that mediates cellular migration have been elucidated, the number of potential targets available to modulate leukocyte trafficking has increased exponentially. In this Viewpoint, we briefly review our current understanding of these mole-cular targets and how these targets vary by tissue and leukocyte subset with emphasis on T cells. We then describe the two currently approved therapeutics that target cell migration, natalizumab and fingolimod, and discuss how an improved understanding of their function could pave the way for the development of safer and more efficacious therapies for inflammatory and autoimmune diseases.

Pure Hemozoin is inflammatory in vivo and activates the NALP3 inflammasome via release of uric acid.

The role of proinflammatory cytokine production in the pathogenesis of malaria is well established, but the identification of the parasite products that initiate inflammation is not complete. Hemozoin is a crystalline metabolite of hemoglobin digestion that is released during malaria infection. In the present study, we characterized the immunostimulatory activity of pure synthetic hemozoin (sHz) in vitro and in vivo. Stimulation of naive murine macrophages with sHz results in the MyD88-independent activation of NF-kappaB and ERK, as well as the release of the chemokine MCP-1; these responses are augmented by IFN-gamma. In macrophages prestimulated with IFN-gamma, sHz also results in a MyD88-dependent release of TNF-alpha. Endothelial cells, which encounter hemozoin after schizont rupture, respond to sHz by releasing IL-6 and the chemokines MCP-1 and IL-8. In vivo, the introduction of sHz into the peritoneal cavity produces an inflammatory response characterized by neutrophil recruitment and the production of MCP-1, KC, IL-6, IL-1alpha, and IL-1beta. MCP-1 and KC are produced independently of MyD88, TLR2/4 and TLR9, and components of the inflammasome; however, neutrophil recruitment, the localized production of IL-1beta, and the increase in circulating IL-6 require MyD88 signaling, the IL-1R pathway, and the inflammasome components ICE (IL-1beta-converting enzyme), ASC (apoptosis-associated, speck-like protein containing CARD), and NALP3. Of note, inflammasome activation by sHz is reduced by allopurinol, which is an inhibitor of uric acid synthesis. These data suggest that uric acid is released during malaria infection and may serve to augment the initial host response to hemozoin via activation of the NALP3 inflammasome.

Toll-like receptor modulation of murine cerebral malaria is dependent on the genetic background of the host.

Infection with Plasmodium berghei ANKA is a well-established model of human cerebral malaria (CM). We show herein that Toll-like receptor (TLR) signaling influences the development of lethal CM in P. berghei ANKA-infected mice. Modulation of outcome was dependent on genetic background, such that deletion of myeloid differentiation factor (MyD) 88 on the susceptible C57BL/6 background resulted in resistance to CM, whereas deletion of MyD88 on the resistant BALB/c background led to increased mortality. Our data show that MyD88 influenced the production of T helper-polarizing cytokines, including interferon (IFN)- gamma, interleukin (IL)-4, and IL-17, as well as the total number of Foxp3(+) regulatory T (T(reg)) cells in a manner dependent on host genetic background. In addition, mRNA levels of IFN- gamma, CXCL10, and CXCL9 were strongly up-regulated in the brains of susceptible wild-type but not MyD88(-/-) infected mice. These results suggest that TLR signaling and host genetic background influences the pathogenesis of CM via modulation of cytokine production and T(reg) cell numbers.

Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo.

Small polybasic peptides derived from the transduction domains of certain proteins, such as the third alpha-helix of the Antennapedia (Antp) homeodomain, can cross the cell membrane through a receptor-independent mechanism. These cell-permeable molecules have been used as 'Trojan horses' to introduce biologically active cargo molecules such as DNA, peptides or proteins into cells. Using these cell-permeable peptides, we have developed an efficient and simple method to increase virally mediated gene delivery and protein expression in vitro and in vivo. Here, we show that cell-permeable peptides increase viral cell entry, improve gene expression at reduced titers of virus and improve efficacy of therapeutically relevant genes in vivo.