Long-Term Outcomes in Patients With Connective Tissue Disease-Associated Pulmonary Arterial Hypertension in the Modern Treatment Era: Meta-Analyses of Randomized, Controlled Trials and Observational Registries.

OBJECTIVE: Data on the magnitude of benefit of modern therapies for pulmonary arterial hypertension (PAH) in connective tissue disease (CTD)-associated PAH are limited. In this study, we performed meta-analyses of randomized, controlled trials (RCTs) and registries to quantify the benefit of these modern therapies in patients with CTD-PAH. METHODS: The PubMed and Embase databases were searched for articles reporting data from RCTs or registries published between January 1, 2000 and November 25, 2019. Eligibility criteria included multicenter studies with ≥30 CTD-PAH patients. For an RCT to be included, the trial had to evaluate an approved PAH therapy, and long-term risks of clinical morbidity and mortality or 6-minute walk distance had to be reported. For a registry to be included, survival rates had to be reported. Random-effects models were used to pool the data. RESULTS: Eleven RCTs (total of 4,329 patients; 1,267 with CTD-PAH) and 19 registries (total of 9,739 patients; 4,008 with CTD-PAH) were included. Investigational therapy resulted in a 36% reduction in the risk of clinical morbidity/mortality events both in the overall PAH population (hazard ratio [HR] 0.64, 95% confidence interval [95% CI] 0.54, 0.75; P < 0.001) and in CTD-PAH patients (HR 0.64, 95% CI 0.51, 0.81; P < 0.001) as compared to control subjects. The survival rate was lower in CTD-PAH patients compared to all PAH patients (survival rate 62%, 95% CI 57, 67% versus 72%, 95% CI 69, 75% at 3 years). The survival rate in CTD-PAH patients treated primarily after 2010 was higher than that in CTD-PAH patients treated before 2010 (survival rate 73%, 95% CI 62, 81% versus 65%, 95% CI 59, 71% at 3 years). CONCLUSION: Modern therapy provides a similar reduction in morbidity/mortality risk in patients with CTD-PAH when compared to the PAH population overall. Risk of death is higher in CTD-PAH patients than in those with PAH overall, but survival has improved in the last 10 years, which may be related to increased screening and/or new treatment approaches. Early detection of PAH in patients with CTD and up-front intensive treatment are warranted.

Human IL-32 expression protects mice against a hypervirulent strain of Mycobacterium tuberculosis.

Silencing of interleukin-32 (IL-32) in a differentiated human promonocytic cell line impairs killing of Mycobacterium tuberculosis (MTB) but the role of IL-32 in vivo against MTB remains unknown. To study the effects of IL-32 in vivo, a transgenic mouse was generated in which the human IL-32γ gene is expressed using the surfactant protein C promoter (SPC-IL-32γTg). Wild-type and SPC-IL-32γTg mice were infected with a low-dose aerosol of a hypervirulent strain of MTB (W-Beijing HN878). At 30 and 60 d after infection, the transgenic mice had 66% and 85% fewer MTB in the lungs and 49% and 68% fewer MTB in the spleens, respectively; the transgenic mice also exhibited greater survival. Increased numbers of host-protective innate and adaptive immune cells were present in SPC-IL-32γTg mice, including tumor necrosis factor-alpha (TNFα) positive lung macrophages and dendritic cells, and IFN-gamma (IFNγ) and TNFα positive CD4(+) and CD8(+) T cells in the lungs and mediastinal lymph nodes. Alveolar macrophages from transgenic mice infected with MTB ex vivo had reduced bacterial burden and increased colocalization of green fluorescent protein-labeled MTB with lysosomes. Furthermore, mouse macrophages made to express IL-32γ but not the splice variant IL-32ß were better able to limit MTB growth than macrophages capable of producing both. The lungs of patients with tuberculosis showed increased IL-32 expression, particularly in macrophages of granulomas and airway epithelial cells but also B cells and T cells. We conclude that IL-32γ enhances host immunity to MTB.

A common polymorphism in extracellular superoxide dismutase affects cardiopulmonary disease risk by altering protein distribution.

BACKGROUND: The enzyme extracellular superoxide dismutase (EC-SOD; SOD3) is a major antioxidant defense in lung and vasculature. A nonsynonomous single-nucleotide polymorphism in EC-SOD (rs1799895) leads to an arginine to glycine amino acid substitution at position 213 (R213G) in the heparin-binding domain. In recent human genetic association studies, this single-nucleotide polymorphism attenuates the risk of lung disease, yet paradoxically increases the risk of cardiovascular disease. METHODS AND RESULTS: Capitalizing on the complete sequence homology between human and mouse in the heparin-binding domain, we created an analogous R213G single-nucleotide polymorphism knockin mouse. The R213G single-nucleotide polymorphism did not change enzyme activity, but shifted the distribution of EC-SOD from lung and vascular tissue to extracellular fluid (eg, bronchoalveolar lavage fluid and plasma). This shift reduces susceptibility to lung disease (lipopolysaccharide-induced lung injury) and increases susceptibility to cardiopulmonary disease (chronic hypoxic pulmonary hypertension). CONCLUSIONS: We conclude that EC-SOD provides optimal protection when localized to the compartment subjected to extracellular oxidative stress: thus, the redistribution of EC-SOD from the lung and pulmonary circulation to the extracellular fluids is beneficial in alveolar lung disease but detrimental in pulmonary vascular disease. These findings account for the discrepant risk associated with R213G in humans with lung diseases compared with cardiovascular diseases.

The cellular distribution of extracellular superoxide dismutase in macrophages is altered by cellular activation but unaffected by the naturally occurring R213G substitution.

Extracellular superoxide dismutase (EC-SOD) is responsible for the dismutation of the superoxide radical produced in the extracellular space and known to be expressed by inflammatory cells, including macrophages and neutrophils. Here we show that EC-SOD is produced by resting macrophages and associated with the cell surface via the extracellular matrix (ECM)-binding region. Upon cellular activation induced by lipopolysaccharide, EC-SOD is relocated and detected both in the cell culture medium and in lipid raft structures. Although the secreted material presented a significantly reduced ligand-binding capacity, this could not be correlated to proteolytic removal of the ECM-binding region, because the integrity of the material recovered from the medium was comparable to that of the cell surface-associated protein. The naturally occurring R213G amino acid substitution located in the ECM-binding region of EC-SOD is known to affect the binding characteristics of the protein. However, the analysis of macrophages expressing R213G EC-SOD did not present evidence of an altered cellular distribution. Our results suggest that EC-SOD plays a dynamic role in the inflammatory response mounted by activated macrophages.

Assessment of airway hyperresponsiveness in mouse models of allergic lung disease using detailed measurements of respiratory mechanics.

This chapter provides an outline of the procedures necessary to measure airway hyperresponsiveness to inhaled methacholine in mouse models of allergic lung disease. We present a method for acquiring detailed measurements of respiratory mechanics using broadband low-frequency oscillatory waveforms applied at the subject's airway opening and analyzed using the constant phase model of the lung. We acknowledge that there are other methods of measuring airway responsiveness in allergic rodent models. However, a discussion of the merits and or detriments of these various methods have been vigorously debated in the primary literature and are beyond the scope of this chapter. The goal of this chapter is to provide a guide in how to begin these types of assays in laboratories which have little to no experience with these particular types of assessments.

The influence of Arhgef1 on pulmonary leukocyte function.

Resident leukocytes in the lungs of healthy individuals are necessary for the innate and adaptive immune response toward potentially harmful foreign antigens that are inhaled on a constant basis. Under normal circumstances, inflammatory stimuli are effectively eradicated via innate immunity with accompanying resolution of inflammation and repair of the lung tissue. Work in our lab has explored how Arhgef1, an intracellular signaling molecule expressed by hematopoietic cells, participates in immune function with an emphasis on its contribution to pulmonary immunity. This review summarizes our studies on the role of Arhgef1 in regulating pulmonary macrophage function.

A novel function of MUC18: amplification of lung inflammation during bacterial infection.

Bacterial infection plays a critical role in exacerbations of various lung diseases, including chronic pulmonary obstructive disease (COPD) and asthma. Excessive lung inflammation is a prominent feature in disease exacerbations, but the underlying mechanisms remain poorly understood. Cell surface glycoprotein MUC18 (alias CD146 or melanoma cell adhesion molecule) has been shown to promote metastasis in several tumors, including melanoma. We explored the function of MUC18 in lung inflammatory responses to bacteria (eg, Mycoplasma pneumoniae) involved in lung disease exacerbations. MUC18 expression was increased in alveolar macrophages from lungs of COPD and asthma patients, compared with normal healthy human subjects. Mouse alveolar macrophages also express MUC18. After M. pneumoniae lung infection, Muc18(-/-) mice exhibited lower levels of the lung proinflammatory cytokines KC and TNF-α and less neutrophil recruitment than Muc18(+/+) mice. Alveolar macrophages from Muc18(-/-) mice produced less KC than those from Muc18(+/+) mice. In Muc18(-/-) mouse alveolar macrophages, adenovirus-mediated MUC18 gene transfer increased KC production. MUC18 amplified proinflammatory responses in alveolar macrophages, in part through enhancing the activation of nuclear factor-κB (NF-κB). Our results demonstrate, for the first time, that MUC18 exerts a proinflammatory function during lung bacterial infection. Up-regulated MUC18 expression in lungs (eg, in alveolar macrophages) of COPD and asthma patients may contribute to excessive inflammation during disease exacerbations.

Sub-chronic exposure to second hand smoke induces airspace leukocyte infiltration and decreased lung elastance.

Exposure to second hand tobacco smoke is associated with the development and/or exacerbation of several different pulmonary diseases in humans. To better understand the possible effects of second hand smoke exposure in humans, we sub-chronically (4 weeks) exposed mice to a mixture of mainstream and sidestream tobacco smoke at concentrations similar to second hand smoke exposure in humans. The inflammatory response to smoke exposures was assessed at the end of this time by enumeration of pulmonary leukocyte infiltration together with measurements of lung elastance and pathology. This response was measured in both healthy wild type (C57BL/6) mice as well as mouse mutants deficient in the expression of Arhgef1 (Arhgef1(-/-)) that display constitutive pulmonary inflammation and decreased lung elastance reminiscent of emphysema. The results from this study show that sub-chronic second hand smoke exposure leads to significantly increased numbers of airspace leukocytes in both healthy and mutant animals. While sub-chronic cigarette smoke exposure is not sufficient to induce changes in lung architecture as measured by mean linear intercept, both groups exhibit a significant decrease in lung elastance. Together these data demonstrate that even sub-chronic exposure to second hand smoke is sufficient to induce pulmonary inflammation and decrease lung elastance in both healthy and diseased animals and in the absence of tissue destruction.

Thromboxane receptor signaling is required for fibronectin-induced matrix metalloproteinase 9 production by human and murine macrophages and is attenuated by the Arhgef1 molecule.

During an inflammatory response, resident and newly recruited tissue macrophages adhere to extracellular matrix and cell-bound integrin ligands. This interaction induces the expression of pro-inflammatory mediators that include matrix metalloproteinases (MMPs). Arhgef1 is an intracellular signaling molecule expressed by myeloid cells that normally attenuates murine macrophage MMP production in vivo and in vitro after cell culture on the extracellular matrix protein, fibronectin. In this study, we have extended the characterization of this fibronectin-induced Arhgef1-regulated signaling pathway in both human and murine myeloid cells. Our results show that MMP9 production by fibronectin-stimulated monocytes and macrophages depends on autocrine thromboxane receptor signaling and that under normal conditions, this signaling pathway is attenuated by Arhgef1. Finally, we show that the expression of ARHGEF1 by human peripheral blood monocytes varies between individuals and inversely correlates with fibronectin-mediated MMP9 production.

Arhgef1 regulates alpha5beta1 integrin-mediated matrix metalloproteinase expression and is required for homeostatic lung immunity.

Pulmonary immunity depends on the ability of leukocytes to neutralize potentially harmful and frequent insults to the lung, and appropriate regulation of leukocyte migration and adhesion is integral to this process. Arhgef1 is a hematopoietic-restricted signaling molecule that regulates leukocyte migration and integrin-mediated adhesion. To explore a possible regulatory role for Arhgef1 in pulmonary immunity we examined the lung and its leukocytes in wild-type and Arhgef1-deficient animals. Here we report that the lungs of Arhgef1-/- mice harbored significantly more leukocytes, increased expression and activity of matrix metalloproteinases (MMPs), airspace enlargement, and decreased lung elastance compared with wild-type lungs. Transfer of Arhgef1-/- lung leukocytes to wild-type mice led to airspace enlargement and impaired lung function, indicating that loss of Arhgef1 in leukocytes was sufficient to induce pulmonary pathology. Furthermore, we showed that Arhgef1-deficient peritoneal macrophages when either injected into the lungs of wild-type mice or cultured on fibronectin significantly increased expression and activity of MMPs relative to control macrophages, and the in vitro fibronectin induction was dependent on the alpha5beta1 integrin pair. Together these data demonstrate that Arhgef1 regulates alpha5beta1-mediated MMP expression by macrophages and that loss of Arhgef1 by leukocytes leads to pulmonary pathology.

Cooperation between mast cells and neurons is essential for antigen-mediated bronchoconstriction.

Mast cells are important sentinels guarding the interface between the environment and the body: a breach in the integrity of this interface can lead to the release of a plethora of mediators that engage the foreign agent, recruit leukocytes, and initiate adaptive physiological changes in the organism. While these capabilities make mast cells critical players in immune defense, it also makes them important contributors to the pathogenesis of diseases such as asthma. Mast cell mediators induce dramatic changes in smooth muscle physiology, and the expression of receptors for these factors by smooth muscle suggests that they act directly to initiate constriction. Contrary to this view, we show herein that mast cell-mediated bronchoconstriction is observed only in animals with intact innervation of the lung and that serotonin release alone is required for this action. While ablation of sensory neurons does not limit bronchoconstriction, constriction after Ag challenge is absent in mice in which the cholinergic pathways are compromised. Linking mast cell function to the cholinergic system likely provides an important means of modulating the function of these resident immune cells to physiology of the lung, but may also provide a safeguard against life-threatening anaphylaxis during mast cell degranulation.

CD38 plays a dual role in allergen-induced airway hyperresponsiveness.

The multifunctional surface protein CD38 acts as a receptor with ecto-enzymatic activity, hydrolyzing NAD to generate several products known to exhibit Ca2+-mobilizing properties. Although CD38 is a convenient marker of immune cell development, and an indicator of progression for several diseases, it is not restricted to the immune compartment. To determine the potentially multilayered involvement of CD38 in allergen-induced airway inflammation and hyperreactivity, we dissected the potential role of CD38 as a regulator of immunity, but also pulmonary function. CD38-deficient and wild-type (WT) mice were sensitized and airway challenged with ovalbumin, and subsequently analyzed regarding their level of airway hyperresponsiveness (AHR) in response to methacholine. Parameters of lung inflammation were also analyzed. Similar sets of measurements were obtained from reciprocal bone marrow swapping experiments between CD38(-/-) and WT mice. Mice lacking CD38 exhibit strongly reduced AHR, which is accompanied by a decrease in typical hallmarks of pulmonary inflammation, including eosinophilia and lymphocytic lung infiltrates, as well as Th2-cytokine levels (IL-4, -5, and -13). Antigen-specific immunoglobulin (Ig)E and IgG1 antibody titers are substantially reduced, consistent with CD38 being crucial for mounting a primary humoral systemic immune response. Reconstitution of lethally irradiated, lung-shielded, CD38-deficient mice with WT bone marrow does not restore WT levels of airway hyperreactivity, nor mucus secretion. The opposite experiment, transferring CD38(-/-) bone marrow into WT mice, also shows reduced AHR levels. These studies demonstrate that CD38 not only acts as a key modulator of the immune response, but also plays an equally important role as an intrinsic pulmonary component.

COX-2-derived prostacyclin protects against bleomycin-induced pulmonary fibrosis.

Prostacyclin is one of a number of lipid mediators elaborated from the metabolism of arachidonic acid by the cyclooxygenase (COX) enzymes. This prostanoid is a potent inhibitor of platelet aggregation, and its production by endothelial cells and protective role in the vasculature are well established. In contrast, much less is known regarding the function of this prostanoid in other disease processes. We show here that COX-2-dependent production of prostacyclin plays an important role in the development of fibrotic lung disease, limiting both the development of fibrosis and the consequential alterations in lung mechanics. In stark contrast, loss of prostaglandin E(2) synthesis and signaling through the G(s)-coupled EP2 and EP4 receptors had no effect on the development of disease. These findings suggest that prostacyclin analogs will protect against bleomycin-induced pulmonary fibrosis in COX-2(-/-) mice. If such protection is observed, investigation of these agents as a novel therapeutic approach to pulmonary fibrosis in humans may be warranted.

Prostaglandin E2 protects lower airways against bronchoconstriction.

Prostaglandin E2 (PGE2), similar to beta-adrenergic receptor agonists, can protect airways from bronchoconstriction and resulting increase in airway resistance induced by a number of agents, including cholinergic receptor agonists and antigen. We examined the impact of sustained alterations in PGE2 pathways on changes in airway resistance. Genetic methods were utilized to alter PGE2 metabolism and signal transduction in the murine lung. PGE2 levels were elevated by generating mice lacking 15-hydroxyprostaglandin (Hpgd-/-), the major catabolic enzyme of PGE2, and by generating a transgenic line in which mouse PGE2 synthase (Ptges) expression is driven by a human lung-specific promoter, hSP-C. Conversely, to determine the impact of loss of PGE2 on airway reactivity, we examined mice lacking this synthase (Ptges-/-) and receptors that mediate the actions of PGE2, particularly the PGE2 EP2 receptor (Ptger2). Diminished capacity to produce and respond to PGE2 did not alter the response of mice to cholinergic stimuli. In contrast, the responsiveness to cholinergic stimulation was dramatically altered in animals with elevated PGE2 levels. The Hpgd-/- and hSP-C-Ptges transgenic lines both showed attenuated airway responsiveness to methacholine as measured by lung resistance. Thus, whereas compromise of the Ptges/PGE2/Ptger2 pathway does not alter airway responsiveness, genetic modulation that elevates PGE2 levels in the lung attenuates airway responsiveness.

Thromboxane A2 induces airway constriction through an M3 muscarinic acetylcholine receptor-dependent mechanism.

Thromboxane A2 (TXA2) is a potent lipid mediator released by platelets and inflammatory cells and is capable of inducing vasoconstriction and bronchoconstriction. In the airways, it has been postulated that TXA2 causes airway constriction by direct activation of thromboxane prostanoid (TP) receptors on airway smooth muscle cells. Here we demonstrate that although TXA2 can mediate a dramatic increase in airway smooth muscle constriction and lung resistance, this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor (mAChR) antagonists. Further analyses employing pharmacological and genetic strategies demonstrate that TP-dependent changes in lung resistance and airway smooth muscle tension require expression of the M2 mAChR subtype. These results raise the possibility that some of the beneficial actions of anticholinergic agents used in the treatment of asthma and chronic obstructive pulmonary disease result from limiting physiological changes mediated through the TP receptor. Furthermore, these findings demonstrate a unique pathway for TP regulation of homeostatic mechanisms in the airway and suggest a paradigm for the role of TXA2 in other organ systems.

Receptors and pathways mediating the effects of prostaglandin E2 on airway tone.

Prostaglandin E(2) (PGE(2)) has complex effects on airway tone, and the existence of four PGE(2) [E-prostanoid (EP)] receptors, each with distinct signaling characteristics, has provided a possible explanation for the seemingly contradictory actions of this lipid mediator. To identify the receptors mediating the actions of PGE(2) on bronchomotor tone, we examined its effects on the airways of wild-type and EP receptor-deficient mice. In conscious mice the administration of PGE(2) increased airway responsiveness primarily through the EP1 receptor, although on certain genetic backgrounds a contribution of the EP3 receptor was detected. These effects of PGE(2) were eliminated by pretreatment with either atropine or bupivacaine and were undetectable in anesthetized mice or in denervated tracheal rings, where only EP2-mediated relaxation of airway smooth muscle was observed. Together, our findings are consistent with a model in which PGE(2) modulates airway tone by activating multiple receptors expressed on various cell populations and in which the relative contribution of these receptors might depend on the expression of modifier alleles. PGE(2)/EP1/EP3-induced airway constriction occurs indirectly through activation of neural pathways, whereas PGE(2)-induced bronchodilation results from direct activation of EP2 receptors on airway smooth muscle. This segregation of EP receptor function within the airway suggests that PGE(2) analogs that selectively activate the EP2 receptor without activating the EP1/EP3 receptors might prove useful in the treatment of asthma.