IL10-driven STAT3 signalling in senescent macrophages promotes pathological eye angiogenesis

Macrophage dysfunction plays a pivotal role during neovascular proliferation in diseases of ageing including cancers, atherosclerosis and blinding eye disease. In the eye, choroidal neovascularization (CNV) causes blindness in patients with age-related macular degeneration (AMD). Here we report that increased IL10, not IL4 or IL13, in senescent eyes activates STAT3 signalling that induces the alternative activation of macrophages and vascular proliferation. Targeted inhibition of both IL10 receptor-mediated signalling and STAT3 activation in macrophages reverses the ageing phenotype. In addition, adoptive transfer of STAT3-deficient macrophages into eyes of old mice significantly reduces the amount of CNV. Systemic and CD163(+) eye macrophages obtained from AMD patients also demonstrate STAT3 activation. Our studies demonstrate that impaired SOCS3 feedback leads to permissive IL10/STAT3 signalling that promotes alternative macrophage activation and pathological neovascularization. These findings have significant implications for our understanding of the pathobiology of age-associated diseases and may guide targeted immunotherapy

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Relation between lysyl-tRNA-synthetase and calreticulin in the context of immunogenic tumor cell death

En réponse aux inducteurs de la mort cellulaire immunogénique, la calréticuline (CRT) est transportée depuis sa localisation orthotopique dans la lumière du réticulum endoplasmique (RE) vers la surface cellulaire où elle va jouer un rôle de signal puissant d’endocytose à l’endroit des cellules présentatrices d’antigène. Ici nous rapportons qu’une autre protéine, la lysyl-ARNt-synthétase (KRS), est exposée à la surface des cellules stressées où elle colocalise avec la CRT au niveau des radeaux lipidiques. La déplétionde KRS à l’aide d’ARNs interférents annihile l’exposition de la CRT induit par les anthracyclines ou les rayonnements UVC. A l’inverse de la CRT, KRS est aussi retrouvée dans le surnageant des cellules stressées. De plus, la protéine KRS recombinante (KRSr) est ici incapable d’influencer la liaison de laCRT recombinante à la surface cellulaire. Et KRSr ne possède pas la capacité de stimuler des macrophages in vitro. Enfin nous révélons que le statut de phosphorylation d’eIF2α constitue un critère différentiel entre l’oxaliplatine et la cisplatine, capable de rendre compte de l’incapacité de cette dernière à entrainer l’exposition de la CRT, et ce malgré une très grande ressemblance chimique entre ces deux composés. Ces résultats mettent donc en exergue la contribution respective de KRS et du stress du RE dans l’émission du signal prototypique de la mort immunogénique.In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopiclocalization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as a potent engulfment signal for antigen-presenting cells. Here, we report that another protein, the lysyl-tRNA-synthetase (KRS), was exposed on the surface of stressed cells, on which KRS co-localized with CRT in lipid rafts. Depletion of KRS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KRS was also found in the supernatant of stressed cells. Recombinant KRS (rKRS) protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, rKRS protein was unable to stimulate macrophages invitro. Finally, we reveal that the phosphorylation status of eIF2α constitute a differential criteria between oxaliplatin and cisplatin (CDDP), accounting for the incapacity of CDDP to trigger the exposure ofCRT, despite their structural and chemical similarities. These results underscore the respective contribution of KRS and ER stress to the emission of the prototypic signal of immunogenic cell death

Impact of human sequences in variable domains of therapeutic antibodies on the location of CD4 T cell epitopes

Although humanization of therapeutic antibodies is widely used to mitigate their immunogenicity, the impact of humanization on T cell response to human(ized) antibodies remains largely unknown. We therefore characterized the peptide specificity of T cells raised against adalimumab (Adm), a human anti-TNF- antibody and natalizumab (Ntz), a humanized anti-integrin α4β1 antibody, both antibodies being immunogenic in patients. Adm-specific T cell response was limited to three regions (HCDR2, HCDR3 and LCDR2) and mainly targeted the HCDR3, which exhibited a broad HLA-DR binding specificity. In contrast, Ntz-specific T cell epitopes resided in HCDR1, HCDR2, HFR3, LCDR1 and LCDR2 but not in HCDR3 demonstrating the uniqueness of a T cell response against therapeutic antibodies. Peptide identification (MAPPs) from HLA-DR molecules of dendritic cells loaded with the antibodies revealed multiple length variants of the presented T cell epitopes across the different donors. Most of the T cell epitopes carry mutations with respect to the germline sequences indicating their potential role for immunogenicity. These data provide molecular determinants involved in the immunogenicity of Adm and Ntz and shed more light on the immunogenicity of human(ized) antibodies in general

Deletion of the fission yeast homologue of human insulinase reveals a TORC1-dependent pathway mediating resistance to proteotoxic stress.

Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+) promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway

Avaliação da sensibilidade oral em crianças com oclusão normal e maloclusão na dentição mista e início da permanente

O objetivo deste trabalho foi avaliar a sensibilidade oral por meio do índice de estereognose oral (hos) em crianças de ambos os sexos, entre 8 e 12 anos de idade com normoclusão ou maloclusão. Para tanto, foram selecionadas 50 crianças, de ambos os sexos, nas fases da dentição mista e início da dentição permanente. Após avaliação clínica as crianças foram divididas em grupos de acordo com as características morfológicas da oclusão: oclusão normal ou maloclusão. A sensibilidade oral foi avaliada pelo índice de estereognose oral, no qual utilizamos peças testes que compreenderam 10 formatos de figuras (círculos, semicírculos, quadrados, retângulos e triângulos em tamanhos pequeno e grande (12x12x3 mm, 8x8x2 mm, respectivamente). Os sujeitos foram classificados quanto ao padrão facial pela fotometria. As variáveis foram analisadas através de estatística descritiva e quantitativa (análise multivariada), e, então, correlacionadas através dos coeficientes de Pearson ou Spearman. As variáveis qualitativas foram associadas às outras variáveis através de testes não paramétricos. Sendo o nível de significância de 0,05% ou p<0,05

Structures of hIDE and model of Iph1 in complex with the insulin B chain.

<p><i>A</i>) Crystallographic structure of the human IDE-E111Q–insulin B chain complex (PDB code 2G56) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067705#pone.0067705-Shen1" target="_blank">[48]</a>. Domains 1, 2, 3 and 4 are colored green, blue, yellow and red, respectively. The Zn²⁺ ion and insulin B chain are colored magenta and orange, respectively. <i>B</i>) Model of an insulin B chain/Iph1 complex. The four domains of Iph1 are drawn in the same orientation and color codes as in <i>A</i>. The two Cys conserved in hIDE are in yellow, the remaining Cys residues are in cyan.</p

Sequence alignment of<i>S.</i><i>pombe</i> Iph1 and hIDE.

<p>Identities are box-shaded in blue and similarities in grey. Non-conserved Cys residues are shaded in yellow and conserved Cys residues in magenta. Residues important for catalysis are box-shaded as follows: residues required for Zn²⁺ coordination, cyan; Glu residue involved in catalysis, red (E71 in Iph1 and E111 in hIDE); E71/E111 were substituted by Asp in strains <i>iph1-E71D</i> and <i>hIDE-E111D</i>. Residues important for substrate recognition and fixation are shaded in green and those required for interaction between the catalytic and the substrate binding domains are shaded in black <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067705#pone.0067705-Shen1" target="_blank">[48]</a>.</p

Lack of Iph1 protects cells from ER stress.

<p><i>A</i>) Drop test of <i>wt</i> and <i>iph1-d</i> strains grown in the indicated medium and exposed or not to TU for 45 min. <i>B</i>) Survival of the indicated strains to TU. The mean (+/− S.E.M) of seven independent experiments is shown. Mann Whitney test was used for statistical analysis. <i>C</i>) Survival to 1 h treatment with different concentrations of TU of <i>wt</i>, <i>iph1-d</i> and protease mutant <i>iph1-E71D</i>. For all strains the mean (+/− S.E.M.) of three independent experiments is shown. ANOVA with Tukey's test was applied for statistical analysis.</p