Third and Fourth Vaccine Doses Broaden and Prolong Immunity to SARS-CoV-2 in Adult Patients with Immune-Mediated Inflammatory Diseases.

Previous studies have reported impaired humoral responses after SARS-CoV-2 mRNA vaccination in patients with immune-mediated inflammatory diseases (IMIDs), particularly those treated with anti-TNF biologics. We previously reported that IMID patients diagnosed with inflammatory bowel disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, or rheumatoid arthritis exhibited greater waning of Ab and T cell responses than healthy control subjects after SARS-CoV-2 vaccine dose 2. Fewer data are available on the effects of third and fourth doses. This observational cohort study collected plasma and PBMCs from healthy control subjects and untreated or treated patients with IMIDs prevaccination and after one to four doses of SARS-CoV-2 mRNA vaccine (BNT162b2 or mRNA-1273). SARS-CoV-2-specific Ab levels, neutralization, and T cell cytokine release were measured against wild-type and Omicron BA.1 and BA.5 variants of concern. Third vaccine doses substantially restored and prolonged Ab and T cell responses in patients with IMIDs and broadened responses against variants of concern. Fourth-dose effects were subtle but also prolonged Ab responses. However, patients with IMIDs treated with anti-TNF, especially patients with inflammatory bowel disease, exhibited lower Ab responses even after the fourth dose. Although T cell IFN-γ responses were maximal after one dose, IL-2 and IL-4 production increased with successive doses, and early production of these cytokines was predictive of neutralization responses at 3-4 mo postvaccination. Our study demonstrates that third and fourth doses of the SARS-CoV-2 mRNA vaccines sustain and broaden immune responses to SARS-CoV-2, supporting the recommendation for three- and four-dose vaccination regimens in patients with IMIDs.

Accelerated waning of immunity to SARS-CoV-2 mRNA vaccines in patients with immune-mediated inflammatory diseases.

BACKGROUNDLimited information is available on the impact of immunosuppressants on COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMID).METHODSThis observational cohort study examined the immunogenicity of SARS-CoV-2 mRNA vaccines in adult patients with inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, or psoriatic disease, with or without maintenance immunosuppressive therapies. Ab and T cell responses to SARS-CoV-2, including neutralization against SARS-CoV-2 variants, were determined before and after 1 and 2 vaccine doses.RESULTSWe prospectively followed 150 subjects, 26 healthy controls, 9 patients with IMID on no treatment, 44 on anti-TNF, 16 on anti-TNF with methotrexate/azathioprine (MTX/AZA), 10 on anti-IL-23, 28 on anti-IL-12/23, 9 on anti-IL-17, and 8 on MTX/AZA. Ab and T cell responses to SARS-CoV-2 were detected in all participants, increasing from dose 1 to dose 2 and declining 3 months later, with greater attrition in patients with IMID compared with healthy controls. Ab levels and neutralization efficacy against variants of concern were substantially lower in anti-TNF-treated patients than in healthy controls and were undetectable against Omicron by 3 months after dose 2.CONCLUSIONSOur findings support the need for a third dose of the mRNA vaccine and for continued monitoring of immunity in these patient groups.FUNDINGFunded by a donation from Juan and Stefania Speck and by Canadian Institutes of Health (CIHR)/COVID-Immunity Task Force (CITF) grants VR-1 172711 and VS1-175545 (to THW and ACG), CIHR FDN-143250 (to THW), GA2-177716 (to VC, ACG, and THW), and GA1-177703 (to ACG) and the CIHR rapid response network to SARS-CoV-2 variants, CoVaRR-Net (to ACG).

Loss of caveolin-1 gene expression accelerates the development of dysplastic mammary lesions in tumor-prone transgenic mice.

Caveolin-1 is the principal structural component of caveolae microdomains, which represent a subcompartment of the plasma membrane. Several independent lines of evidence support the notion that caveolin-1 functions as a suppressor of cell transformation. For example, the human CAV-1 gene maps to a suspected tumor suppressor locus (D7S522/7q31.1) that is frequently deleted in a number of carcinomas, including breast cancers. In addition, up to 16% of human breast cancers harbor a dominant-negative mutation, P132L, in the CAV-1 gene. Despite these genetic associations, the tumor suppressor role of caveolin-1 still remains controversial. To directly assess the in vivo transformation suppressor activity of the caveolin-1 gene, we interbred Cav-1 (-/-) null mice with tumor-prone transgenic mice (MMTV-PyMT) that normally develop multifocal dysplastic lesions throughout the entire mammary tree. Herein, we show that loss of caveolin-1 gene expression dramatically accelerates the development of these multifocal dysplastic mammary lesions. At 3 wk of age, loss of caveolin-1 resulted in an approximately twofold increase in the number of lesions (foci per gland; 3.3 +/- 1.0 vs. 7.0 +/- 1.2) and an approximately five- to sixfold increase in the total area occupied by these lesions. Similar results were obtained at 4 wk of age. However, complete loss of caveolin-1 was required to accelerate the appearance of these dysplastic mammary lesions, because Cav-1 (+/-) heterozygous mice did not show any increases in foci development. We also show that loss of caveolin-1 increases the extent and the histological grade of these mammary lesions and facilitates the development of papillary projections in the mammary ducts. Finally, we demonstrate that cyclin D1 expression levels are dramatically elevated in Cav-1 (-/-) null mammary lesions, consistent with the accelerated appearance and growth of these dysplastic foci. This is the first in vivo demonstration that caveolin-1 can function as a transformation suppressor gene.

Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression.

OBJECTIVES: Pulmonary hypertension (PH) and lung structural remodeling are frequent complications of congestive heart failure (CHF). Yet, the molecular mechanisms involved in CHF-induced PH and lung remodeling remain unknown. Caveolins (Cav-1, -2 and -3) are the principal structural proteins of the vesicular invaginations of the plasma membrane, termed caveolae. Mice with homozygous deletion of the caveolin-1 gene (Cav-1(-/-)) have been shown to develop dilated cardiomyopathy, PH and lung structural remodeling, characterized by hypercellularity and thickening of the alveolar septa. However, the physiological relevance of these observations for the pathogenesis of PH and lung remodeling remains to be determined. METHODS AND RESULTS: Here, we investigate the natural behavior of the endogenous caveolin proteins during the development of PH and lung structural remodeling, using a rat model of myocardial infarction (MI). MI was induced in male Wistar rats by ligating the left anterior coronary artery. Two weeks post-MI, rats were anesthetized and hemodynamic and morphometric measurements were obtained. Rats subjected to MI developed marked PH, lung structural remodeling and right ventricular hypertrophy (RVH). Both immunoblot analysis and immunohistochemistry dramatically show that Cav-1 and Cav-2 expression is downregulated to almost undetectable levels in the lungs of post-MI rats. Mechanistically, the reduced expression of caveolins was associated with the increased tyrosine-phosphorylation of the signal transducer and activator of transcription-3 (STAT3) and the upregulation of cyclin D1 and D3 expression. We also show that STAT3 is hyperphosphorylated, and cyclin D1 and D3 levels are dramatically upregulated, in lung tissue samples derived from Cav-1 (-/-)- and Cav-2 (-/-)-deficient mice. CONCLUSIONS: Thus, down-modulation of pulmonary Cav-1 and Cav-2 expression in rats subjected to MI may represent an initiating mechanism leading to the activation of the STAT3/Cyclins pathway and, ultimately, to the development of PH and lung structural remodeling.

Role of caveolin-1 in the regulation of lipoprotein metabolism.

Lipoprotein metabolism plays an important role in the development of several human diseases, including coronary artery disease and the metabolic syndrome. A good comprehension of the factors that regulate the metabolism of the various lipoproteins is therefore key to better understanding the variables associated with the development of these diseases. Among the players identified are regulators such as caveolins and caveolae. Caveolae are small plasma membrane invaginations that are observed in terminally differentiated cells. Their most important protein marker, caveolin-1, has been shown to play a key role in the regulation of several cellular signaling pathways and in the regulation of plasma lipoprotein metabolism. In the present paper, we have examined the role of caveolin-1 in lipoprotein metabolism using caveolin-1-deficient (Cav-1(-/-)) mice. Our data show that, while Cav-1(-/-) mice show increased plasma triglyceride levels, they also display reduced hepatic very low-density lipoprotein (VLDL) secretion. Additionally, we also found that a caveolin-1 deficiency is associated with an increase in high-density lipoprotein (HDL), and these HDL particles are enriched in cholesteryl ester in Cav-1(-/-) mice when compared with HDL obtained from wild-type mice. Finally, our data suggest that a caveolin-1 deficiency prevents the transcytosis of LDL across endothelial cells, and therefore, that caveolin-1 may be implicated in the regulation of plasma LDL levels. Taken together, our studies suggest that caveolin-1 plays an important role in the regulation of lipoprotein metabolism by controlling their plasma levels as well as their lipid composition. Thus caveolin-1 may also play an important role in the development of atherosclerosis.

Caveolin-1 and regulation of cellular cholesterol homeostasis.

Caveolae are 50- to 100-nm cell surface plasma membrane invaginations present in terminally differentiated cells. They are characterized by the presence of caveolin-1, sphingolipids, and cholesterol. Caveolin-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. We have recently generated caveolin-1-deficient [Cav-1(-/-)] mice in which caveolae organelles are completely eliminated from all cell types, except cardiac and skeletal muscle. In the present study, we examined the metabolism of cholesterol in wild-type (WT) and Cav-1(-/-) mouse embryonic fibroblasts (MEFs) and mouse peritoneal macrophages (MPMs). We observed that Cav-1(-/-) MEFs are enriched in esterified cholesterol but depleted of free cholesterol compared with their wild-type counterparts. Similarly, Cav-1(-/-) MPMs also contained less free cholesterol and were enriched in esterified cholesterol on cholesterol loading. In agreement with this finding, caveolin-1 deficiency was associated with reduced free cholesterol synthesis but increased acyl-CoA:cholesterol acyl-transferase (ACAT) activity. In wild-type MPMs, we observed that caveolin-1 was markedly upregulated on cholesterol loading. Despite these differences, cellular cholesterol efflux from MEFs and MPMs to HDL was not affected in the Cav-1-deficient cells. Neither ATP-binding cassette transporter G1 (ABCG1)- nor scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux was affected. Cellular cholesterol efflux to apolipoprotein A-I was not significantly reduced in Cav-1(-/-) MPMs compared with wild-type MPMs. However, ABCA1-mediated cholesterol efflux was clearly more sensitive to the inhibitory effects of glyburide in Cav-1(-/-) MPMs versus WT MPMs. Taken together, these findings suggest that caveolin-1 plays an important role in the regulation of intracellular cholesterol homeostasis and can modulate the activity of other proteins that are involved in the regulation of intracellular cholesterol homeostasis.

Muscle-specific interaction of caveolin isoforms: differential complex formation between caveolins in fibroblastic vs. muscle cells.

It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-deficient mouse embryonic fibroblasts, in which we have stably expressed caveolin-1, -2, or -3, we find that caveolin-1 interacts with caveolin-2 in this setting, whereas caveolin-3 does not, in agreement with most published observations. Next, we used a transfected L6 myoblast cell system expressing all three caveolin proteins. Surprisingly, we found that caveolin-1, -2, and -3 all coimmunoprecipitate in this cell type, suggesting that this interaction is muscle cell specific. Similar results were obtained when the skeletal muscle of caveolin-1 transgenic animals was analyzed for caveolin-1 and caveolin-3 coimmunoprecipitation. Thus we conclude that all three caveolins can interact to form a discrete hetero-oligomeric complex, but that such complex formation is clearly muscle specific.

Combined loss of INK4a and caveolin-1 synergistically enhances cell proliferation and oncogene-induced tumorigenesis: role of INK4a/CAV-1 in mammary epithelial cell hyperplasia.

Tumorigenesis is a multistep process that involves a series of genetic changes or "multiple hits," leading to alterations in signaling, proliferation, immortalization, and transformation. Many of the molecular factors that govern tumor initiation and progression remain unknown. Here, we evaluate the transformation suppressor potential of caveolin-1 (Cav-1) and its ability to cooperate with a well established tumor suppressor, the INK4a locus. To study the effects of loss of caveolin-1 on cellular transformation, we established immortalized primary mouse embryonic fibroblasts (MEFs) expressing and lacking caveolin-1 by interbreeding Cav-1 (+/+) and Cav-1 (-/-) mice with INK4a (-/-) mice. Analysis of these cells reveals that loss of caveolin-1 confers a significant growth advantage, as measured via cellular proliferation and cell cycle analysis. Loss of caveolin-1 in the INK4a (-/-) genetic background results in constitutive hyperactivation of the p42/44 MAP kinase cascade, decreased expression of p21(Cip1), as well as cyclin D1 and PCNA overexpression, consistent with their hyperproliferative phenotype. Importantly, in cells lacking Cav-1 expression, transformation by activated oncogenes (H-Ras(G12V) or v-Src) results in increased tumor growth in vivo (up to >40-fold). Finally, INK4a (-/-)/Cav-1 (-/-) mice demonstrate disturbed mammary epithelial ductal morphology, with hyperplasia, increased side-branching, and fibrosis. Our results provide important new evidence for the transformation suppressor properties of Cav-1 and the first molecular genetic evidence that Cav-1 cooperates with a tumor suppressor, namely the INK4a genetic locus.

Urogenital alterations in aged male caveolin-1 knockout mice.

PURPOSE: Caveolae are flask-shaped invaginations of the plasma membrane formed by the oligomerization of caveolins. Because only smooth muscle contains all caveolin (Cav) family members (Cav-1, 2 and 3), we examined the contribution of each caveolin to urogenital smooth muscle structure/function. MATERIALS AND METHODS: WT, Cav-1, 2, 3 and -1/3 knockout (KO) mouse bladders were characterized by Western blot, co-immunoprecipitation, immunofluorescence microscopy, electron microscopy, histochemistry and pharmacological techniques. Cystometric analysis was performed in conscious, freely moving mice. Other urogenital organs were investigated by histological analysis. RESULTS: The loss of bladder Cav-1 results in a marked decrease in Cav-2 but not Cav-3 expression. Ablation of Cav-3 fails to alter Cav-1 or Cav-2 expression. Deletion of Cav-1 results in the almost complete loss of caveolae, while Cav-2 KO and Cav-3 KO mouse smooth muscle showed a normal number of caveolae. The loss of Cav-1 generated caveolae led to significant urogenital changes in male mice (most marked by 12 months of age), namely 1) bladder weight-to-body weight ratios were increased, 2) the bladder smooth muscle layer was thickened, 3) the bladders had increased baseline, threshold and spontaneous pressures, 4) bladder strips showed a decreased contractile response to carbachol and KCl, and 5) these smooth muscle changes were accompanied by marked fluid accumulation in the prostate and seminal vesicles, with intracellular vacuolization in the kidneys. As such, male Cav-1 KO mice may be a useful animal model for studying LUTD (lower urinary tract dysfunction) that is so prevalent in aging male patients. CONCLUSIONS: The loss of Cav-1 and, thus, of most smooth muscle cell caveolae results in significant bladder dysfunction and urogenital organ changes in aged male mice.

Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade.

A growing body of evidence suggests that muscle cell caveolae may function as specialized membrane micro-domains in which the dystrophin-glycoprotein complex and cellular signaling molecules reside. Caveolin-3 (Cav-3) is the only caveolin family member expressed in striated muscle cell types (cardiac and skeletal). Interestingly, skeletal muscle fibers from Cav-3 (-/-) knock-out mice show a number of myopathic changes, consistent with a mild-to-moderate muscular dystrophy phenotype. However, it remains unknown whether a loss of Cav-3 affects the phenotypic behavior cardiac myocytes in vivo. Here, we present a detailed characterization of the hearts of Cav-3 knock-out mice. We show that these mice develop a progressive cardiomyopathic phenotype. At four months of age, Cav-3 knock-out hearts display significant hypertrophy, dilation, and reduced fractional shortening, as revealed by gated cardiac MRI and transthoracic echocardiography. Histological analysis reveals marked cardiac myocyte hypertrophy, with accompanying cellular infiltrates and progressive interstitial/peri-vascular fibrosis. Interestingly, loss of Cav-3 expression in the heart does not change the expression or the membrane association of the dystrophin-glycoprotein (DG) complex. However, a marker of the DG complex, alpha-sarcoglycan, was specifically excluded from lipid raft domains in the absence of Cav-3. Because activation of the Ras-p42/44 MAPK pathway in cardiac myocytes can drive cardiac hypertrophy, we next assessed the activation state of this pathway using a phospho-specific antibody probe. We show that p42/44 MAPK (ERK1/2) is hyperactivated in hearts derived from Cav-3 knock-out mice. These results are consistent with previous in vitro data demonstrating that caveolins may function as negative regulators of the p42/44 MAPK cascade. Taken together, our data argue that loss of Cav-3 expression is sufficient to induce a molecular program leading to cardiac myocyte hypertrophy and cardiomyopathy.