Martin V. Richter

A muscle resident cell population promotes fibrosis in hindlimb skeletal muscles of mdx mice through the Wnt canonical pathway

Previous work has pointed to a role for the Wnt canonical pathway in fibrosis formation in aged skeletal muscles. In the present study, we studied the dystrophic mdx mouse, which displays skeletal muscle fibrosis. Our results indicated that the muscle resident stromal cell (mrSC) population in the muscles of dystrophic mice is higher than in the muscles of age-matched wild-type mice. Wnt3a promoted the proliferation of and collagen expression by cultured mrSCs but arrested the growth of and collagen expression by cultured myoblasts. Injections of Wnt3A in the tibialis anterior muscles of adult wild-type mice significantly enhanced the mrSC population and collagen deposition compared with the contralateral muscles. Conversely, an injection of the Wnt antagonist Dickkof protein (DKK1) into the skeletal muscles of mdx mice significantly reduced collagen deposition. These results suggested that the Wnt canonical pathway expands the population of mrSCs and stimulates their production of collagen as observed during aging and in various myopathies.

Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

ABSTRACT Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract

Factors from the virus and the host contribute to influenza virus pathogenicity and to the development of immunity. This study thoroughly examined the effects of an initial infectious dose of virus and unveiled new findings concerning the antiviral and inflammatory responses, innate and adaptive immunity, memory responses, and protection against secondary heterologous infection. Our results demonstrated that the initial infectious dose significantly affects the gene expression of antiviral (IFN‐β) and inflammatory (TNF‐α, IL‐6, IL‐1β) cytokines and of enzymes involved in nitrosative/oxidative stress (iNOS, HO‐1, NQO1) early in the response to influenza. This response correlated with significantly increased recruitment of innate immune cells into the lungs of infected mice. We showed that this response also alters the subsequent accumulation of activated IFN‐γ+ CD44hi CD62Llo influenza‐specific CD8+ T cells into the lungs of infected mice through increased T cell‐recruiting chemokine gene expression (CCL3, CCL4, CCL5, CXCL10). Furthermore, we demonstrated that the initial infectious dose determines the generation and the distribution of memory CD8+ T cell subsets without affecting trafficking mechanisms. This impacted on immune protection against heterologous infection. Lastly, we showed that the effects on innate and adaptive immunity were not dependent on influenza strain or on the genetic background of the host. Collectively, our data show for the first time and in detail that the initial infectious dose of influenza determines the development of several aspects of antiviral immunity. This study provides new insights on virus‐host interaction in the generation of the global immune response to influenza.

The Prostanoid 15-Deoxy-Δ12,14-Prostaglandin-J2 Reduces Lung Inflammation and Protects Mice Against Lethal Influenza Infection

BACKGROUND Growing evidence indicates that influenza pathogenicity relates to altered immune responses and hypercytokinemia. Therefore, dampening the excessive inflammatory response induced after infection might reduce influenza morbidity and mortality. METHODS Considering this, we investigated the effect of the anti-inflammatory molecule 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) in a mouse model of lethal influenza infection. RESULTS Administration of 15d-PGJ(2) on day 1 after infection, but not on day 0, protected 79% of mice against lethal influenza infection. In addition, this treatment considerably reduced the morbidity associated with severe influenza infection. Our results also showed that treatment with 15d-PGJ(2) decreased influenza-induced lung inflammation, as shown by the diminished gene expression of several proinflammatory cytokines and chemokines. Unexpectedly, 15d-PGJ(2) also markedly reduced the viral load in the lungs of infected mice. This could be attributed to maintained type I interferon gene expression levels after treatment. Interestingly, pretreatment of mice with a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist before 15d-PGJ(2) administration completely abrogated its protective effect against influenza infection. CONCLUSIONS Our results demonstrate for the first time that treatment of mice with 15d-PGJ(2) reduces influenza morbidity and mortality through activation of the PPARγ pathway. PPARγ agonists could thus represent a potential therapeutic avenue for influenza infections.

Prospective heterotopic ossification progenitors in adult human skeletal muscle

Skeletal muscle has strong regenerative capabilities. However, failed regeneration can lead to complications where aberrant tissue forms as is the case with heterotopic ossification (HO), in which chondrocytes, osteoblasts and white and brown adipocytes can arise following severe trauma. In humans, the various HO cell types likely originate from multipotent mesenchymal stromal cells (MSCs) in skeletal muscle, which have not been identified in humans until now. In the present study, adherent cells from freshly digested skeletal muscle tissue were expanded in defined culture medium and were FACS-enriched for the CD73(+)CD105(+)CD90(-) population, which displayed robust multilineage potential. Clonal differentiation assays confirmed that all three lineages originated from a single multipotent progenitor. In addition to differentiating into typical HO lineages, human muscle resident MSCs (hmrMSCs) also differentiated into brown adipocytes expressing uncoupling protein 1 (UCP1). Characterizing this novel multipotent hmrMSC population with a brown adipocyte differentiation capacity has enhanced our understanding of the contribution of non-myogenic progenitor cells to regeneration and aberrant tissue formation in human skeletal muscle.

Agreement of bioelectric impedance analysis and dual-energy X-ray absorptiometry for body composition evaluation in adults with cystic fibrosis

Malnutrition in cystic fibrosis (CF) is associated with increased mortality and can lead to fat-free (FFM) and fat mass (FM) loss. Dual-energy X-ray absorptiometry (DXA) is used and validated to measure FFM and FM. DXAs high cost has led to the utilization of less costly techniques such as bioelectrical impedance analysis (BIA). The aim of this study was to determine the agreement of FFM, FM and %FM measurements taken with DXA and BIA in adults with CF. We measured FFM, FM and %FM in 34 adults with CF with a leg-to-leg BIA and an iDXA and determined agreement using Bland-Altman analysis. While DXA and BIA measurements were well correlated (r > 0.8), mean biases between both methods were between 8 and 11%. BIA underestimated FM and %FM and overestimated FFM. In a clinical research setting where these measurements are used to phenotype patients, BIA cannot replace DXA.

Could T cells be involved in lung deterioration and hyperglycemia in cystic fibrosis

Cystic fibrosis-related diabetes (CFRD) is the most frequent complication of cystic fibrosis (CF) and associated with increased mortality. Why patients have an accelerated loss of lung function before the diagnosis of CFRD remains poorly understood. We reported that patients with or without CFRD had increased glucose excursions when compared to healthy peers. Studies have demonstrated that patients with CF have increased glucose fluctuations and hyperglycemia and that this may affect the clinical course of CF and lead to lymphocyte dysfunction. T-helper 17 (Th17) lymphocytes produce and secrete the pro-inflammatory cytokine IL-17. The Th17 pathway is involved in CF lung inflammation, β-cell destruction in type 1 diabetes (T1D) and Th17 cells of patients with type 2 diabetes have increased production of IL-17 when compared to healthy peers. Also, regulatory T-cells (Tregs) have been shown to be dysfunctional and produce IL-17 in T1D. Furthermore, vitamin D can affect inflammation in CF, diabetes and the differentiation of lymphocytes. In this review, we discuss the potential roles of hyperglycemia on Th17 cells, Tregs and IL-17 as a potential cause for accelerated lung function decline before CFRD and how this could be modulated by vitamin D or by directly intervening in the IL-17A pathway.

The administration of oseltamivir results in reduced effector and memory CD8+ T cell responses to influenza and affects protective immunity

The clinical benefits of oseltamivir (Tamiflu) are well established, but the effects of antiviral treatment on the immune response are poorly understood. By use of flow cytometric analyses and the mouse model, we thoroughly investigated the impact of such a treatment on the immune response and the generation of protective immunity to influenza. We demonstrated that influenza‐specific CD8+ effector T cell recruitment was reduced up to 81% in the lungs of mice treated with oseltamivir (5 or 50 mg/kg twice daily; EC50 49 nM in vitro) compared to saline controls, but cell generation was unaffected in draining lymph nodes. Importantly, we showed that oseltamivir administration significantly decreased the pools of tissue‐resident and circulating effector memory (93.7%) and central memory CD8+ T cells (45%) compared to saline controls. During heterologous secondary infection, a decreased memory CD8+ T cell pool combined with reduced generation of secondary influenza‐specific effectors in the lymph nodes resulted in 10‐fold decreased CD8+ T cell recall responses, which increased mouse morbidity and delayed viral clearance. Furthermore, antiviral administration led to a significant 5.7‐fold decreased production of functional anti‐influenza antibodies. Thus, our study demonstrates that antiviral treatment affects the development of the adaptive immune response and protective immunity against influenza.—Marois, I., Cloutier, A., Garneau, É., Lesur, O., Richter, M. V., The administration of oseltamivir results in reduced effector and memory CD8+ T cell responses to influenza and affects protective immunity. FASEB J. 29, 973–987 (2015). www.fasebj.org

The Flavonoid Isoliquiritigenin Reduces Lung Inflammation and Mouse Morbidity during Influenza Virus Infection

ABSTRACT The host response to influenza virus infection is characterized by an acute lung inflammatory response in which intense inflammatory cell recruitment, hypercytokinemia, and a high level of oxidative stress are present. The sum of these events contributes to the virus-induced lung damage that leads to high a level of morbidity and mortality in susceptible infected patients. In this context, we identified compounds that can simultaneously reduce the excessive inflammatory response and the viral replication as a strategy to treat influenza virus infection. We investigated the anti-inflammatory and antiviral potential activities of isoliquiritigenin (ILG). Interestingly, we demonstrated that ILG is a potent inhibitor of influenza virus replication in human bronchial epithelial cells (50% effective concentration [EC50] = 24.7 μM). In addition, our results showed that this molecule inhibits the expression of inflammatory cytokines induced after the infection of cells with influenza virus. We demonstrated that the anti-inflammatory activity of ILG in the context of influenza virus infection is dependent on the activation of the peroxisome proliferator-activated receptor gamma pathway. Interestingly, ILG phosphate (ILG-p)-treated mice displayed decreased lung inflammation as depicted by reduced cytokine gene expression and inflammatory cell recruitment. We also demonstrated that influenza virus-specific CD8+ effector T cell recruitment was reduced up to 60% in the lungs of mice treated with ILG-p (10 mg/kg) compared to that in saline-treated mice. Finally, we showed that administration of ILG-p reduced lung viral titers and morbidity of mice infected with the PR8/H1N1 virus.

Instability of Trinucleotidic Repeats During Chromatin Remodeling in Spermatids

Transient DNA breaks and evidence of DNA damage response have recently been reported during the chromatin remodeling process in haploid spermatids, creating a potential window of enhanced genetic instability. We used flow cytometry to achieve separation of differentiating spermatids into four highly purified populations using transgenic mice harboring 160 CAG repeats within exon 1 of the human Huntington disease gene (HTT). Trinucleotic repeat expansion was found to occur immediately following the chromatin remodeling steps, confirming the genetic instability of the process and pointing to the origin of paternal anticipation observed in some trinucleotidic repeats diseases.