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Phagocytosis is an important leucocyte function, however using existing models it cannot be measured in human tissues in vivo. To address this, we characterized a new phagocytosis model using intradermal methylene blue-labelled Escherichia coli injection (MBEC). Methylene blue (MB) is a licensed human medicine and bacterial stain potentially useful for labelling E. coli that are safe for human injection. Ex vivo co-culture of leucocytes with MBEC caused MB to transfer into neutrophils and macrophages by phagocytosis. During this, a 'red shift' in MB fluorescence was shown to be caused by phagolysosomal oxidisation. Hence, MBEC co-culture could be used to measure phagocytosis and phagolysosomal oxidisation in humans, ex vivo. In healthy volunteers, inflammatory exudate sampling using suction blisters 2-24h after intradermal MBEC injection showed that tissue-acquired neutrophils and monocytes contained more MB than their circulating counterparts, whereas blood and inflamed tissue T, B and NK cells were MBlo. This was validated with spectral flow cytometry by visualizing the MB emission spectrum in tissue-acquired neutrophils. Neutrophil MB emission spectra demonstrated more 'red shift' at 24h compared to earlier time-points, in-keeping with progressive phagolysosomal MB oxidisation in neutrophils over time in vivo. This new MBEC model can therefore measure bacterial phagocytosis and phagolysosomal oxidisation in human skin, in vivo. This has a number of important research applications, for example in studying human phagocyte biology, testing novel antimicrobials, and understanding why certain groups such as males, the elderly or those with diabetes, recent surgery or malnutrition are at increased risk of bacterial infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE 2 ) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4 + /CD44 + /CD62L + and CD4 + /CD44 + /CD62L - /CD27 + T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE 2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury., (© 2024. The Author(s).)

Background: Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinical oncology research method. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD- scid IL2Rgamma null (NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin., Methods: The immunophenotype of lymphoproliferations arising in the lung TRACERx PDX pipeline were characterised. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview)., Results: Lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma with plasma cell features. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins., Discussion: Overall, these data suggest that B cell clones with lymphoproliferative potential are present within primary NSCLC tumours, and that these are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines., Competing Interests: CS acknowledges grant support from AstraZeneca, Boehringer-Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae previously Archer Dx Inc - collaboration in minimal residual disease sequencing technologies, and Ono Pharmaceutical. CS is an AstraZeneca Advisory Board member and Chief Investigator for the AZ MeRmaiD 1 and 2 clinical trials and is also Co-Chief Investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s Scientific Advisory Board. CS receives consultant fees from Achilles Therapeutics also SAB member, Bicycle Therapeutics also a SAB member, Genentech, Medicxi, Roche Innovation Centre - Shanghai, Metabomed until July 2022, and the Sarah Cannon Research Institute. CS has received honoraria from Amgen, AstraZeneca, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, Illumina, and Roche-Ventana. CS had stock options in Apogen Biotechnologies and GRAIL until June 2021, and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and has stock options and is co-founder of Achilles Therapeutics. CS holds patents relating to assay technology to detect tumour recurrence PCT/GB2017/053289; to targeting neoantigens PCT/EP2016/059401, identifying patent response to immune checkpoint blockade PCT/EP2016/071471, determining HLA LOH PCT/GB2018/052004, predicting survival rates of patients with cancer PCT/GB2020/050221, identifying patients who respond to cancer treatment PCT/GB2018/051912, US patent relating to detecting tumour mutations PCT/US2017/28013, methods for lung cancer detection US20190106751A1 and both a European and US patent related to identifying insertion/deletion mutation targets PCT/GB2018/051892. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pearce, Akarca, De Maeyer, Kostina, Huebner, Sivakumar, Karasaki, Shah, Janes, McGranahan, Reddy, Akbar, Moore, Marafioti, Swanton and Hynds.)

Background & Aims: Infection is a major problem in advanced liver disease secondary to monocyte dysfunction. Elevated prostaglandin (PG)E 2 is a mediator of monocyte dysfunction in cirrhosis; thus, we examined PGE 2 signalling in outpatients with ascites and in patients hospitalised with acute decompensation to identify potential therapeutic targets aimed at improving monocyte dysfunction., Methods: Using samples from 11 outpatients with ascites and 28 patients hospitalised with decompensated cirrhosis, we assayed plasma levels of PGE 2 and lipopolysaccharide (LPS); performed quantitative real-time PCR on monocytes; and examined peripheral blood monocyte function. We performed western blotting and immunohistochemistry for PG biosynthetic machinery expression in liver tissue. Finally, we investigated the effect of PGE 2 antagonists in whole blood using polychromatic flow cytometry and cytokine production., Results: We show that hepatic production of PGE 2 via the cyclo-oxygenase 1-microsomal PGE synthase 1 pathway, and circulating monocytes contributes to increased plasma PGE 2 in decompensated cirrhosis. Transjugular intrahepatic sampling did not reveal whether hepatic or monocytic production was larger. Blood monocyte numbers increased, whereas individual monocyte function decreased as patients progressed from outpatients with ascites to patients hospitalised with acute decompensation, as assessed by Human Leukocyte Antigen (HLA)-DR isotype expression and tumour necrosis factor alpha and IL6 production. PGE 2 mediated this dysfunction via its EP 4 receptor., Conclusions: PGE 2 mediates monocyte dysfunction in decompensated cirrhosis via its EP 4 receptor and dysfunction was worse in hospitalised patients compared with outpatients with ascites. Our study identifies a potential drug target and therapeutic opportunity in these outpatients with ascites to reverse this process to prevent infection and hospital admission., Lay Summary: Patients with decompensated cirrhosis (jaundice, fluid build-up, confusion, and vomiting blood) have high infection rates that lead to high mortality rates. A white blood cell subset, monocytes, function poorly in these patients, which is a key factor underlying their sensitivity to infection. We show that monocyte dysfunction in decompensated cirrhosis is mediated by a lipid hormone in the blood, prostaglandin E 2 , which is present at elevated levels, via its EP 4 pathway. This dysfunction worsens when patients are hospitalised with complications of cirrhosis compared with those in the outpatients setting, which supports the EP 4 pathway as a potential therapeutic target for patients to prevent infection and hospitalisation., Competing Interests: None of the authors have any conflict of interests or disclosures relevant to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2021 The Author(s).)

While COVID-19, the disease driven by SARS-CoV-2 has ignited interest in the host immune response to this infection, it has also highlighted the lack of treatment options for the damaging inflammatory responses driven by pathogens that precipitate the acute respiratory distress syndrome (ARDS). With the global prevalence of SARS-CoV-2 and the likelihood of a second winter spike alongside seasonal flu, the need for effective and targeted anti-inflammatory agents is even more pressing. Here we discuss the aetiology of COVID-19 and the common signalling pathways driven by SARS-CoV-2, namely p38 MAP kinase. We highlight that p38 MAP kinase becomes elevated with increasing age, thereby driving many of the inflammatory pathways that precipitate death in old people with the added drawback of impairing vaccine efficacy in this susceptible age group. Finally, we review drugs available to inhibit p38 MAP kinase, their risks-versus-benefits as well as suggested dosing regimen to combat over-exuberant innate immune responses and potentially reverse vaccine inefficacy in older patients., Competing Interests: Declaration of Competing Interest DWG and MT have equity ownership in Senex Therapeutics Inc. MU is an employee of AstraZeneca and holds share in the company. The other authors declare no competing interests., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Ageing is a global burden. Increasing age is associated with increased incidence of infections and cancer and decreased vaccine efficacy. This increased morbidity observed with age, is believed to be due in part to a decline in adaptive immunity, termed immunosenescence. However not all aspects of immunity decrease with age as ageing presents with systemic low grade chronic inflammation, characterised by elevated concentrations of mediators such as IL-6, TNFα and C Reactive protein (CRP). Inflammation is a strong predictor of morbidity and mortality, and chronic inflammation is known to be detrimental to a functioning immune system. Although the source of the inflammation is much discussed, the key cells which are believed to facilitate the inflammageing phenomenon are the monocytes and macrophages. In this review we detail how macrophage and monocyte phenotype and function change with age. The impact of ageing on macrophages includes decreased phagocytosis and immune resolution, increased senescent-associated markers, increased inflammatory cytokine production, reduced autophagy, and a decrease in TLR expression. With monocytes there is an increase in circulating CD16 + monocytes, decreased type I IFN production, and decreased efferocytosis. In conclusion, we believe that monocytes and macrophages contribute to immunosenescence and inflammageing and as a result have an important role in defective immunity with age., (Copyright © 2020 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

The development of senescence in tissues of different organs and in the immune system are usually investigated independently of each other although during ageing, senescence in both cellular systems develop concurrently. Senescent T cells are highly inflammatory and secrete cytotoxic mediators and express natural killer cells receptors (NKR) that bypass their antigen specificity. Instead they recognize stress ligands that are induced by inflammation or infection of different cell types in tissues. In this article we discuss data on T cell senescence, how it is regulated and evidence for novel functional attributes of senescent T cells. We discuss an interactive loop between senescent T cells and senescent non-lymphoid cells and conclude that in situations of intense inflammation, senescent cells may damage healthy tissue. While the example for immunopathology induced by senescent cells that we highlight is cutaneous leishmaniasis, this situation of organ damage may apply to other infections, including COVID-19 and also rheumatoid arthritis, where ageing, inflammation and senescent cells are all part of the same equation., (© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Increasing age alters innate immune-mediated responses; however, the mechanisms underpinning these changes in humans are not fully understood. Using a human dermal model of acute inflammation, we found that, although inflammatory onset is similar between young and elderly individuals, the resolution phase was substantially impaired in elderly individuals. This arose from a reduction in T cell immunoglobulin mucin receptor-4 (TIM-4), a phosphatidylserine receptor expressed on macrophages that enables the engulfment of apoptotic bodies, so-called efferocytosis. Reduced TIM-4 in elderly individuals was caused by an elevation in macrophage p38 mitogen-activated protein kinase (MAPK) activity. Administering an orally active p38 inhibitor to elderly individuals rescued TIM-4 expression, cleared apoptotic bodies and restored a macrophage resolution phenotype. Thus, inhibiting p38 in elderly individuals rejuvenated their resolution response to be more similar to that of younger people. This is the first resolution defect identified in humans that has been successfully reversed, thereby highlighting the tractability of targeting pro-resolution biology to treat diseases driven by chronic inflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Aging is associated with remodeling of the immune system to enable the maintenance of life-long immunity. In the CD8 + T cell compartment, aging results in the expansion of highly differentiated cells that exhibit characteristics of cellular senescence. Here we found that CD27 - CD28 - CD8 + T cells lost the signaling activity of the T cell antigen receptor (TCR) and expressed a protein complex containing the agonistic natural killer (NK) receptor NKG2D and the NK adaptor molecule DAP12, which promoted cytotoxicity against cells that expressed NKG2D ligands. Immunoprecipitation and imaging cytometry indicated that the NKG2D-DAP12 complex was associated with sestrin 2. The genetic inhibition of sestrin 2 resulted in decreased expression of NKG2D and DAP12 and restored TCR signaling in senescent-like CD27 - CD28 - CD8 + T cells. Therefore, during aging, sestrins induce the reprogramming of non-proliferative senescent-like CD27 - CD28 - CD8 + T cells to acquire a broad-spectrum, innate-like killing activity.

Acute inflammation is characterized by granulocyte infiltration followed by efferocytosing mononuclear phagocytes, which pave the way for inflammatory resolution. Until now, it was believed that resolution then leads back to homeostasis, the physiological state tissues experience before inflammation occurred. However, we discovered that resolution triggered a prolonged phase of immune suppression mediated by prostanoids. Specifically, once inflammation was switched off, natural killer cells, secreting interferon γ (IFNγ), infiltrated the post-inflamed site. IFNγ upregulated microsomal prostaglandin E synthase-1 (mPGES-1) alongside cyclo-oxygenase (COX-1) within macrophage populations, resulting in sustained prostaglandin (PG)E 2 biosynthesis. Whereas PGE 2 suppressed local innate immunity to bacterial infection, it also inhibited lymphocyte function and generated myeloid-derived suppressor cells, the net effect of which was impaired uptake/presentation of exogenous antigens. Therefore, we have defined a sequence of post-resolution events that dampens the propensity to develop autoimmune responses to endogenous antigens at the cost of local tissue infection., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)