Biology subjects

Graham, G. J.

Publications and source records attributed to Graham, G. J..

10 recordsLinked to original sources

bioRxiv2025

Macrophages drive inguinal fat pad and lymph node remodelling in response to peripheral inflammation.

Adaptive immune responses are intensely energy-dependent and rely on a local source of fuel-producing molecules which have been proposed to be derived from fat pads in which mammalian lymph nodes are embedded. However, the trigger for their release has not been identified. Here we demonstrate that cutaneous inflammation is directly correlated with rapid atrophy of perinodal fat pads and increase in embedded lymph node size. We further demonstrate that the fat pad atrophy is associated with influx of a CCR2-independent, lipid metabolising, macrophage population. Macrophage depletion ameliorates fat pad atrophy, and lymph node expansion, downstream of inflamed sites. Our data therefore identify peripheral inflammation as an antigen-independent trigger of downstream fat pad and lymph node remodelling and contributes to the release of essential nutrients to drive the energetic requirements of the adaptive immune response.

immunology↗

bioRxiv2024

Generation of the First Transgenic Line of the Iconic Coral Reef Fish Amphiprion ocellaris

The common clownfish, Amphiprion ocellaris, is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an Elongation factor 1 (Ef1): Green fluorescent protein (GFP) cassette at various concentrations, needle tip dimensions and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n=10) that were genotyped GFP+ were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP+ (n= 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5 %) produced GFP+ offspring. Further, two F1s crossed with WT females produced 53.8% and 54.2% GFP+ offspring respectively, confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology.

bioengineering↗

bioRxiv2024

Novel Behavioral Assays Reveal Sex Specific Behavioral Syndromes in Anemonefish

Sexually dimorphic behaviors are common across taxa, particularly in the contexts of parental care and territorial aggression. The false clown anemonefish Amphiprion ocellaris is unique among animals for its combination of female behavioral dominance and territoriality, protandrous sex change, and mutualistic symbiosis with sea anemones. Several laboratory studies have begun characterizing sex differences in parental care and aggression in this species, but aggression assays have mostly focused on intra-specific aggression where individual differences are large. The goals of this study were to expand the repertoire of behavioral assays available for A. ocellaris, establish repeatability of individual differences, identify assays that produce the most robust sex differences, and explore whether individual differences in correlated behaviors can be detected consistently across experimental contexts (i.e., whether behavioral syndromes can be detected). To this end, we measured 39 behaviors across 7 behavioral assays (parental care, large intruder aggression, small intruder aggression, male-oriented aggression, female-oriented aggression, immediate reaction to a threat, and nest maintenance) in 9 reproductively active A. ocellaris pairs under 3 different contexts (without eggs in the nest, with their own eggs, and with surrogate eggs). Behaviors were repeatedly measured three separate times (rounds) over repeated spawning cycles. We found 34 out of 39 behaviors were significantly individually repeatable across egg contexts and rounds, with an average intra-class correlation of 0.33. We found parental care, large intruder aggression, and female-oriented aggression assays produced the largest sex differences. Males performed 7-fold more egg care behaviors than females, while females performed 3.5-fold more aggressive behaviors toward a large interspecific intruder. Further, females bit, chased and struck the female intruder 6.5, 4.1, and 3.2 times as many times as males did. Five different behavioral syndromes were observed in males but only one was observed in females. These results expand our understanding of sex differences in behavior and the division of labor in the iconic anemonefish. Future studies can use these assays to measure the behavioral sex of fish in the middle of sex change, in the study of behavioral plasticity, or in the study of the neuroendocrine bases of aggression and parental care in this unique species.

animal behavior and cognition↗

bioRxiv2022

CXCR2 inhibition enables NASH-HCC immunotherapy

ObjectiveHepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DesignNeutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. ResultsNeutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoral XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoral immunity, this was confirmed by loss of therapeutic effect upon genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a pro-tumor to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. ConclusionCXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC. Significance of this studyWhat is already known on this subject? O_LIImmune checkpoint inhibition therapy is emerging as a promising new therapy for the treatment of advanced hepatocellular carcinoma (HCC). C_LIO_LIOnly a minority of HCC patients will respond to immune checkpoint inhibition (ICI) therapy and recent data suggest that HCC on the background of NASH may have reduced sensitivity to this treatment strategy. C_LIO_LINeutrophils are a typical myeloid component of the liver in NASH and are found either within the HCC tumour microenvironment or in a peritumoural location. C_LIO_LINeutrophils have considerable phenotypic plasticity and can exist in both tumour promoting and tumour suppressing states. C_LIO_LINeutrophils may have the ability to influence ICI therapy. C_LI What are the new findings? O_LICXCR2+ neutrophils are found in human NASH and within the tumour of both human and mouse models of NASH-HCC. C_LIO_LIThe resistance of NASH-HCC to anti-PD1 therapy is overcome by co-treatment with a CXCR2 small molecule inhibitor, with evidence of reduced tumour burden and extended survival. C_LIO_LIAnti-PD1 and CXCR2 inhibitor combine to selectively reprogramme tumour-associated neutrophils (TANs) from a pro- to an anti-tumour phenotype. C_LIO_LIReprogrammed TANs proliferate locally within Granzyme B+ immune clusters that contain physically associating CD8+ T cells and antigen presenting cells. C_LIO_LIConventional XCR1+ dendritic cells (cDC1s) are found to be elevated in anti-PD1 and CXCR2 inhibitor treated HCCs and together with CD8+ T cells are required for therapeutic benefit. C_LI How might it impact on clinical practice in the foreseeable future? O_LITANs can be selectively manipulated to adopt an anti-tumour phenotype which unlocks their potential for cancer therapy. The ability of CXCR2 antagonism to combine with ICI therapy to bring about enhanced therapeutic benefit in NASH-HCC (and potentially in HCC of other aetiologies) warrents clinical investigation. C_LI

cancer biology↗

bioRxiv2021

Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice

Inflammatory chemokines and their receptors are central to the development of inflammatory/immune pathologies. The apparent complexity of this system, coupled with lack of appropriate in vivo models, has limited our understanding of how chemokines orchestrate inflammatory responses and has hampered attempts at targeting this system in inflammatory disease. Novel approaches are therefore needed to provide crucial biological, and therapeutic, insights into the chemokine-chemokine receptor family. Here, we report the generation of transgenic multi-chemokine receptor reporter mice in which spectrally-distinct fluorescent reporters mark expression of CCRs 1, 2, 3 and 5, key receptors for myeloid cell recruitment in inflammation. Analysis of these animals has allowed us to define, for the first time, individual and combinatorial receptor expression patterns on myeloid cells in resting and inflamed conditions. Our results demonstrate that chemokine receptor expression is highly specific, and more selective than previously anticipated.

immunology↗

bioRxiv2021

Maturation, developmental site, and pathology dictate murine neutrophil function

Neutrophils have been implicated in poor outcomes in cancer and severe inflammation. We found that neutrophils expressing intermediate levels of Ly6G (Ly6GInt) were present in mouse cancer models and more abundant in those with high rates of spontaneous metastasis. Maturation, age, tissue localization and functional capacity all drive neutrophil heterogeneity. Recent studies have proposed various markers to distinguish between these heterogeneous sub-populations; however, these markers are limited to specific models of inflammation and cancer. Here, we identify and define Ly6G expression level as a robust and reliable marker to distinguish neutrophils at different stages of maturation. Ly6GInt neutrophils were bona fide immature neutrophils with reduced immune regulatory and adhesion capacity. Whereas the bone marrow is a more recognised site of granulopoiesis, the spleen also produces neutrophils in homeostasis and cancer. Strikingly, neutrophils matured faster in the spleen than in the bone marrow with unique transcriptional profiles. We propose that developmental origin is critical in neutrophil identity and postulate that neutrophils that develop in the spleen supplement the bone marrow by providing an intermediate more mature reserve before emergency haematopoiesis.

immunology↗

bioRxiv2020

Diverse myeloid cells are recruited to the developing and inflamed mammary gland

The immune system plays fundamental roles in the mammary gland, shaping developmental processes and controlling inflammation during infection and cancer. Here we reveal unanticipated heterogeneity in the myeloid cell compartment during development of virgin, pregnant and involuting mouse mammary glands, and in milk. We investigate the functional consequences of individual and compound chemokine receptor deficiency on cell recruitment. Diverse myeloid cell recruitment was also shown in models of sterile inflammation and bacterial infection. Strikingly, we have shown that inflammation and infection can alter the abundance of terminal end buds, a key developmental structure, within the pubertal mammary gland. This previously unknown effect of inflammatory burden during puberty could have important implications for understanding the control of pubertal timing.

immunology↗

bioRxiv2019

Sorting for CCR7-positivity enriches for a dendritic cell population with enhanced antigen-presenting capacity and anti-tumour potency

Dendritic cell therapy has been a promising addition to the current armoury of therapeutic options in cancer for more than 20 years but has not yet achieved break-through success. To successfully initiate immunity, dendritic cells have to enter the lymph nodes. However, previous experience of therapeutic dendritic cell administration indicates that this is frequently an extremely inefficient process. The major regulator of dendritic cell migration to the lymph nodes is the chemokine receptor CCR7 and in vitro generated dendritic cells typically display heterogenous expression of this receptor. Here we demonstrate that positive-selection for the dendritic cell subpopulation expressing CCR7 enriches for cells with enhanced lymph node migration and antigen presentation competence as well as a chemokine expression profile indicative of improved interactions with T cells. In models of both subcutaneous and metastatic melanoma we demonstrate that the dendritic cells sorted for CCR7 expression trigger enhanced CD8 T-cell driven antitumour immune responses which correlate with reduced tumour burden and increased survival. Finally we demonstrate that this approach is directly translatable to human dendritic cell therapy using clinical-grade cell sorting.\n\nSynopsisTherapeutic dendritic cells drive anti-cancer immune responses but they migrate inefficiently to lymph nodes. We show that enriching for CCR7 expression yields a dendritic cellular product with enhanced ability to generate immune responses to localised and disseminated tumours.

immunology↗

bioRxiv2019

Pan-viral protection against arboviruses by targeting inoculation site-based skin macrophages

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The large number of genetically-distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made developing virus-specific anti-viral medicines challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies at this site. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical innate immune agonist significantly suppressed both the local and subsequent systemic course of infection and improved clinical outcome in mice to infection with a variety of arboviruses from the Alphavirus, Flavivirus and Orthobunyavirus genuses. In the absence of treatment, anti-viral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, targeting the more populous skin-resident macrophages with an immune agonist elicited protective responses in key cellular targets of virus that otherwise replicated virus to high levels. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have shown that it is possible to improve outcome to infection by targeting pathways activated at the site of inoculation, and thereby identified a putative new strategy for limiting disease following infection with a variety of genetically-distinct arboviruses.\n\nOne-sentence summaryWe demonstrate that activation of innate immune responses to arbovirus at the mosquito bite is a limiting factor for preventing efficient systemic dissemination of virus and that therapeutic targeting of skin-resident macrophages can have defining inhibitory effects on the later systemic course.

immunology↗

bioRxiv2018

The chemokine receptor CXCR2 contributes to murine adipocyte development

Chemokines are members of a large family of chemotactic cytokines that signal through their receptors to mediate leukocyte recruitment during inflammation and homeostasis. The chemokine receptor CXCR2 has largely been associated with neutrophil recruitment. However, there is emerging evidence of roles for chemokines and their receptors in processes other than leukocyte migration. We have previously demonstrated that CXCR2 KO mice have thinner skin compared to wild type mice. Herein we demonstrate that this is due to a thinner subcutaneous adipose layer, as a result of fewer and smaller individual adipocytes. We observe a similar phenotype in other fat depots and present data that suggests this may be due to reduced expression of adipogenesis related genes associated with adipocyte specific CXCR2 signalling. Interestingly, this phenotype is evident in female, but not male, CXCR2 KO mice. These findings expand our understanding of non-leukocyte related chemokine receptor functions and help to explain some previously observed adipose-related phenotypes in CXCR2 KO mice.

immunology↗