Hugo Garneau

E2F4 expression is required for cell cycle progression of normal intestinal crypt cells and colorectal cancer cells.

The generation of knock‐out mice for E2F4 gene expression has suggested a role for this transcription factor in establishing and/or maintaining the intestinal crypt compartment. Having previously demonstrated that E2F4 is cytoplasmic in quiescent‐differentiated cells but nuclear in growth factor‐stimulated proliferative cells, the present study was aimed at determining the role of E2F4 in the control of human intestinal epithelial proliferation. Results herein demonstrate that lentiviral infection of an shRNA which specifically knocked‐down E2F4 expression slowed down G1/S phase transition and the proliferation rate of normal human intestinal epithelial cells (HIEC) and of colon cancer cells. Protein expression of Cdk2, cyclins D1 and A, Cdc25A and c‐myc was markedly down‐regulated in shE2F4‐expressing cells; by contrast, expression of the cell cycle inhibitors p21Cip/Waf and p27Kip1 was increased. In addition, the expression of many genes involved in DNA synthesis was down‐regulated in shE2F4‐expressing cells, whereas no modulation in E2F1 expression was observed. A decrease in E2F4 in colon cancer cell lines also resulted in a reduction in soft‐agar growth capacity. Immunofluorescence experiments in human fetal intestine revealed that cells expressing high nuclear levels of E2F4 also expressed cyclin A protein. Lastly, E2F4 and its target cyclin A were up‐regulated and mostly nuclear in human colorectal tumor cells in comparison to the corresponding benign epithelium. These results indicate that nuclear E2F4 may be determinant in the promotion of proliferation of human intestinal epithelial crypt cells and colorectal cancer cells. J. Cell. Physiol. 221: 350–358, 2009.

Aging, immunosenescence and membrane rafts: the lipid connection

The decreased efficiency of immune responses in older people is partly a consequence of alterations in T lymphocyte functions caused by modifications in the early events of signal transduction. Several alterations in the signaling pathways of T lymphocytes have been described in older humans and animals. A unifying cause could be modifications in the physicochemical properties of the plasma membrane resulting from changes in its lipid composition and the distribution and function of lipid rafts (LR). The latter serve to assemble the initial components of the signaling cascade. Accumulating data suggest that the function of plasma membrane LR is altered with aging; we hypothesize that this would significantly contribute to immune dysregulation. The role of aging and cholesterol in LR functions in T lymphocytes is reviewed and discussed here.

Downregulation of inhibitory SRC Homology 2 Domain-containing Phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly

BackgroundImmune responses are generally impaired in aged mammals. T cells have been extensively studied in this context due to the initial discovery of their reduced proliferative capacity with aging. The decreased responses involve altered signaling events associated with the early steps of T cell activation. The underlying causes of these changes are not fully understood but point to alterations in assembly of the machinery for T cell activation. Here, we have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms that participate in the regulation of the early steps of T cell activation. Such conditions tip the immune balance in favor of altered T cell activation and a related decreased response in aging.ResultsWe present evidence that the tyrosine phosphatase SHP-1, a key regulator of T cell signal transduction machinery is, at least in part, responsible for the impaired T cell activation in aging. We used tyrosine-specific mAbs and Western blot analysis to show that a deregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of tyrosine-phosphorylated Lck (Y505). Confocal microscopy analysis revealed that the dynamic movements of these regulatory proteins in lipid raft microdomains was altered in T cells of aged individuals. Enzymic assays showed that SHP-1 activity was upregulated in T cells of aged donors, in contrast to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels approaching those of young donors. Significant differences in the active (Y394) and inactive (Y505) phosphorylation sites of Lck in response to T cell activation were observed in elderly donors as compared to young subjects, independently of CD45 isoform expression.ConclusionsOur data suggest that the role of SHP-1 in T cell activation extends to its increased effect in negative feedback in aging. Modulation of SHP-1 activity could be a target to restore altered T cell functions in aging. These observations could have far reaching consequences for improvement of immunosenescence and its clinical consequences such as infections, altered response to vaccination.

Predictors of the antibody response to influenza vaccination in older adults with type 2 diabetes.

Objective Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic inflammatory diseases of the elderly. Its development is related to the alteration of the immune system with aging characterized by immunosenescence and inflamm-aging. In turn, T2DM also alters the immune response. As a consequence, older people with T2DM are more susceptible to influenza and to its complications as compared with healthy controls. Vaccination against influenza has shown poor efficacy in the older population and even less efficacy in patients with diabetes. We studied here the antibody response to vaccination in healthy and diabetic elderly participants. Research design and methods In 2 groups of elderly participants (healthy N=119 and T2DM N=102), we measured the immunogenicity of influenza vaccine by hemagglutination inhibition assays. We assessed several blood and functional parameters as potential predictors of the vaccine efficacy. Results We found no difference between antibody responses in diabetic elderly compared with healthy elderly. Among the biological and functional determinants, the cytomegalovirus (CMV) serostatus played a more prominent role in determining the magnitude of response. We concluded that in addition to age and diabetic status, immunological history such as CMV status should be taken into account. None of the other biological or functional parameters studied could be reliably linked to the vaccine antibody response in older adults who are not frail including those with well-controlled diabetes. Conclusions Our data strongly suggest that influenza vaccine should be administered to elderly patients with T2DM; however, the immune determinants of the antibody response to influenza vaccination should be further investigated.

Cytomegalovirus Seropositivity Predicts a Decline in the T Cell But Not the Antibody Response to Influenza in Vaccinated Older Adults Independent of Type 2 Diabetes Status.

Abstract Type 2 diabetes mellitus (T2DM) and persistent cytomegalovirus (CMV) infection are postulated contributors to inflammatory processes that impact on the age-related decline in T-cell responses to influenza vaccination. Older subjects with T2DM (n = 30) and healthy aged controls (n = 40) were enrolled and received influenza vaccination in this study. Serum inflammatory markers and CMV serostatus were measured. Pre- to post-vaccination changes in serum antibody titers to the A/H3N2 strain, and levels of granzyme B (GrB, cytotoxic T lymphocytes) in lysates and cytokines in supernatants from influenza A/H3N2-challenged peripheral blood mononuclear cells were measured. We found no difference between the T2DM and healthy groups in the immune responses measured. However, CMV serostatus was a key determinant of the GrB response to influenza challenge; CMV+ subjects had low levels of inducible GrB (iGrB) activity in response to influenza challenge. In contrast, the serum antibody response to the A/H3N2 vaccine strain did not differ with CMV serostatus, and serum levels of the inflammatory marker, β2-microglobulin, were positively correlated with age, T2DM, and serum IL-10 levels. In conclusion, CMV seropositivity associated with a decline in GrB responses to influenza may predict increased susceptibility to influenza in older adults.

Altered neutrophil functions in elderly patients during a 6-month follow-up period after a hip fracture.

BACKGROUND Fracture of the hip (HF) is a significant cause of morbidity and mortality in elderly individuals. HF is an acute stress that triggers a state of inflammation which may affect immune responses and physical recovery. METHODS Longitudinal study of the impact of HF on the functions of polymorphonuclear neutrophils (PMNs) in elderly subjects. Data were recorded prior to surgery, 6weeks and 6months later. RESULTS PMN functions were severely impaired shortly after HF (chemotaxis, phagocytosis, superoxide production) but there was a time-related recovery of some PMN functions (chemotaxis, phagocytosis) over time, except in the case of superoxide production. Whereas FcγRII (CD32) expression remained unchanged, FcγRIII (CD16) increased from low values before surgery to levels of controls 6months post-surgery. This was also the case for the C5a complement receptor and CD11b. TLR2 and TLR4 expressions were unchanged. Cytokine and chemokine secretions by stimulated PMN were altered. TNFα and IL-10 secretions were increased following HF but IL-8 secretion was decreased. Impaired PMN functions prior to surgery were related to alterations in PI3K and NF-κB signaling pathways. Recovery of these functions paralleled increased PI3K activity, although superoxide production remained low. Sustained activation of the NF-κB pathway by TNFα has been reported to involve upregulation of IKKβ kinase activity. Activated IKKβ kinase inhibits ERK1/2 and results in concomitant downstream inhibition of NADPH oxidase complex which can account for sustained impaired production of ROS in HF patients. CONCLUSION Our data showed that the stress caused by HF negatively affects initial PMN responses shortly after the event and that may negatively influence clinical outcomes such as resolving long-term inflammation and recovery, as well as explaining susceptibility to opportunistic infections.

Stimulation of ENaC Activity by Rosiglitazone is PPARγ-Dependent and Correlates with SGK1 Expression Increase

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopuslaevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.

Differential Phenotypes of Myeloid-Derived Suppressor and T Regulatory Cells and Cytokine Levels in Amnestic Mild Cognitive Impairment Subjects Compared to Mild Alzheimer Diseased Patients

Alzheimer disease (AD) is the most prevalent form of dementia although the underlying cause(s) remains unknown at this time. However, neuroinflammation is believed to play an important role and suspected contributing immune parameters can be revealed in studies comparing patients at the stage of amnestic mild cognitive impairment (aMCI) to healthy age-matched individuals. A network of immune regulatory cells including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) maintains immune homeostasis but there are very few data on the role of these cells in AD. Here, we investigated the presence of these cells in the blood of subjects with aMCI and mild AD (mAD) in comparison with healthy age-matched controls. We also quantitated several pro- and anti-inflammatory cytokines in sera which can influence the development and activation of these cells. We found significantly higher levels of MDSCs and Tregs in aMCI but not in mAD patients, as well as higher serum IL-1β levels. Stratifying the subjects based on CMV serostatus that is known to influence multiple immune parameters showed an absence of differences between aMCI subjects compared to mAD patients and healthy controls. We suggest that the increase in MDSCs and Tregs number in aMCI subjects may have a beneficial role in modulating inflammatory processes. However, this protective mechanism may have failed in mAD patients, allowing progression of the disease. This working hypothesis obviously requires testing in future studies.

Alterations in Monocyte Phenotypes and Functions after a Hip Fracture in Elderly Individuals: A 6-Month Longitudinal Study.

Background: Healthy elderly individuals are particularly prone to catastrophic events at any moment of their lives. One stressful event for individuals aged 65 and older is a fall that results in a fracture of the hip (HF). HF causes a state of inflammation that may affect immune responses. In this connection, we have reported that HF induced alterations in neutrophil functions. Objective: To assess the impact of HF on classical (cM), intermediate (iM) and non-classical (ncM) monocyte subsets. Methods: Distribution, functions (chemotaxis, phagocytosis, superoxide production and cytokine production), phenotype and activation (NF-κB and PI3K) were evaluated in monocyte subsets before surgery and 6 weeks and 6 months after the event. Results: The distribution of cM and ncM was unchanged, but iM transiently increased before surgery. Sustained increases (iM response to CCL2 and CX3CL1) and decreases (cM and ncM response to CCL2) in chemotaxis were observed. Phagocytosis and superoxide production were impaired in cM but not in iM or ncM. Sustained expression of HLA-DR occurred in cM but not in iM and ncM. Sustained decreased expression of CD11b occurred only in ncM. Sustained decreases (cM and ncM) and increases (iM) in CCR2 expression were observed. An elevated expression of CX3CR1 was found only in iM. cM produced elevated quantities of TNFα. There was a transient oxidative burst of production before surgery in iM and a sustained decrease in ncM. IL-10 production was severely impaired in cM and decreased in iM prior to surgery. Sustained activation (cM), inhibition (ncM) and transient activation (iM) of NF-κB were observed. Activation of PI3K was severely impaired in cM and ncM but was sustained in iM. Conclusion: HF had more impact on cM and ncM functions than on iM. HF triggered a switch in cM functions from phagocytic to inflammatory elevated TNFα-producing cells. These changes may impact clinical outcomes of HF with respect to inflammation, opportunistic infections and physical recovery.

Immunological Methods and the Concept of Inflammaging in the Study of Human Aging

Abstract Many theories have been proposed to explain the phenomenon of aging, but the cause of this complex process is still not fully understood. All physiological functions, including immunity, are affected by the aging process; thus, “immunosenescence” is believed to contribute not only to an increased incidence and severity of infectious disease but also to be a major protagonist in the etiology of different chronic diseases with an inflammatory component (the latter dubbed a result of “inflammaging”). Data from many studies suggest that human longevity is influenced by immunosenescence and inflammaging, but cause and effect remain controversial. In this chapter, we will review the age-related differences in immune response parameters in humans and assess methods for studying them in the context of an integrated aging process.