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Tumour-associated macrophages (TAMs) are key immune-suppressive constituents of the breast tumour microenvironment (TME) and correlate with poor prognosis. Tenascin-C (TNC) is an immunomodulatory extracellular matrix (ECM) protein which is upregulated in breast tumours, and is associated with increased metastasis and worsened prognosis. Our previous work showed that perturbation of the breast TME by tumour-derived TNC skews macrophages towards an immune-suppressive phenotype featuring downregulated antigen-presentation. Using TNC as an exemplar perturbant, this thesis sought to understand how macrophage effector functions such as antigen presentation become enhanced or suppressed within the TME. To achieve this, single-cell RNA sequencing (scRNAseq) was performed on infiltrating myeloid cells derived from syngeneic, orthotopically-engrafted, murine breast tumours that were engineered to express high or low levels of TNC. scRNAseq revealed marked heterogeneity in the intra-tumoural myeloid compartment with 7 distinct subsets observed, 5 of which were macrophages (Mac1-5). Notably, macrophages delegated key effector functions including antigen presentation (Mac2) and chemokine production (Mac3) to discrete subsets that were phenotypically, spatially and temporally distinct at the protein level by flow cytometry and confocal immunofluorescence imaging. The role of TNC was predominantly exerted at the level of subset abundance (rather than gene transcription), with 4 out of 7 myeloid subsets exhibiting TNC-dependent changes in abundance, including two macrophage subsets (Mac1 & Mac2). Over time, TNC modestly weakened the segregation of Mac2 and Mac3 phenotypes, suggesting it may drive dysfunctional delegation of effector functions. Pseudotemporal analysis of scRNAseq data implicated the epigenetic regulator, Kdm6b, in governing the acquisition of Mac3 effector function, and pharmacological inhibition of KDM6B in vivo shifted the proportion of Mac3 via TAM reprogramming. Finally, mouse-derived myeloid subsets were well-conserved in the most comprehensive scRNAseq atlas of human breast cancer, and gene signature analyses in bulk clinical datasets demonstrated significant differences in prognosis for patients with distinct myeloid 'ecotype' profiles. Together, these data demonstrate functionally divergent roles for TAM subsets in breast tumour progression and metastasis, while providing mechanistic insights into the impact of ECM-derived cues such as TNC on shaping the myeloid compartment, with potential therapeutic and prognostic implications for breast cancer patients.

Innate myeloid cells, such as macrophages and dendritic cells (DC), regulate the type, magnitude, and duration of the immune response. Macrophages and DCs do so, by instructing the functions of effector cells, such as natural killer (NK) cells. NK cells' effector functions are important in the killing of virus or bacterial-infected and malignantly transformed cells. NK cells also participate in the production of cytokines like IFN-γ, IL-10 and TNF after viral, fungal or bacterial infection. CALHMs (calcium homeostasis modulators) are a family of membrane proteins recently identified as voltage-gated oligomeric ion- and ATP channels. CALHM1 and 3 specifically, form a channel that localises in the synapse between taste bud cells and neurons, which it is important for the formation of a synaptic structure. Interestingly, a single member of the CALHM family, CALHM6 is expressed almost exclusively in immune cells. Like other CALHMs, CALHM6 forms a higher order complex, composed of up to 11 oligomers, with a channel-like structure. Previous studies show that CALHM6 is required for the instruction of antitumour effector responses in NK cells, but its role in infections and its mechanism of action remains unknown. In this work, we show that in myeloid cells, CALHM6 is highly induced on myeloid cells in response to pathogen-derived signals and is inhibited by anti-inflammatory cytokines. Also, while the structure of CALHM6 resembles a higher oligomeric version of the CALHM1/3 channel, we show that its gating is different as it does not release ATP in response to the same stimuli that open CALHM1/3. To understand the function of CALHM6, we generated CALHM6 deficient mice (Calhm6-/-). To test whether CALHM6 plays a role in the immune response to infections we challenged WT and Calhm6-/- mice with Listeria monocytogenes (L. monocytogenes) an intracellular gram-positive bacterium that elicits a strong innate immune response and requires NK cells derived IFN-γ for its clearance by activated macrophages. Calhm6-/- mice fail to control L. monocytogenes burden at peak infection. This is not caused by an intrinsic failure of macrophages in killing Listeria but it is likely driven by an early deficiency in NK-derived IFN-γ, which causes a delay in the kinetic of the innate immune response. It is well appreciated that the most efficient activation of NK cells in vivo requires their direct priming/interaction with activated macrophages/DC. Calhm6-/- mice are specifically unable to activate NK cells in a DC- and macrophage-dependent manner. Bone marrow-derived macrophages from Calhm6-/- mice fail to upregulate mRNA expression of synaptic-associated genes when stimulated with pro-inflammatory stimuli. This together with our findings that CALHM6 relocates to the macrophage-NK cell interface in vitro, suggests that CALHM6 localises in the immunological synapse between innate and NK cells, where it forms a channel that is important in the innate cell-mediated instruction of NK cell activation.

Neutrophils are important effector cells in innate immunity, possessing a wide range of effector functions, including reactive oxygen species (ROS) production, phagocytosis and chemotaxis. These properties enable neutrophils to rapidly respond to stimulation and orchestrate protective immunity. Currently, the transcriptional regulatory networks underlying neutrophil activation and function remain largely unexplored. Of note, there is partial understanding of transcription factors (TFs) that act as key regulators to control neutrophil development and inflammatory responses. Neutrophils undergo tightly controlled genomic and transcriptional changes while transitioning between bone marrow, blood, and tissue sites. However, the molecular mechanisms underlining neutrophil function during inflammation have yet to be fully elucidated. To determine the transcription factor networks that shape these responses, we have undertaken integrated transcriptional and chromatin analyses of neutrophils during acute inflammation and revealed distinct sets of putative transcription factors (TFs) associated with control of neutrophil differentiation and inflammatory responses. To investigate the regulatory role of the selected TFs in neutrophils, I utilised HoxB8 myeloid progenitors as a model system for in vitro production of neutrophils and generated stable knockout lines for JUNB, RELB, IRF5, RFX2, KLF6, and RUNX1 in Hoxb8 myeloid progenitors, using the CRISPR/Cas9 mediated system. Additionally, I have demonstrated the importance of RUNX1, KLF6 in neutrophil differentiation. RUNX1, KLF6 deletion in HoxB8 progenitors caused a block in neutrophil differentiation and produced lower levels of mature neutrophils. KLF6 and RUNX1 deficient neutrophils also displayed impaired transmigration properties. Consistent with findings in the Hoxb8 in vitro model, conditional deletion of RUNX1 in myeloid populations produced lower mature neutrophils in the bone marrow. To validate the functional role of the selected TFs in neutrophil activation, I examined the consequence of CEBPβ, RELB, IRF5, JUNB knockouts on neutrophil effector functions, such as phagocytosis, cytokine production, generation of ROS, formation of NETs and bacterial killing. I found that RELB, IRF5, JUNB knockout significantly affect the ability of neutrophils to produce inflammatory mediators. JUNB and RELB also contribute to neutrophil phagocytosis, ROS generation and NETosis.

Interferon regulatory factor 5 (IRF5), is a multifunctional regulator of immune responses, with a critical role in controlling pro-inflammatory responses of macrophages. IRF5 contributes to the pathogenesis of several inflammatory and autoimmune diseases. Given the importance of IRF5 function in physiology and disease, IRF5 represents a therapeutic target. However, the mechanisms regulating IRF5 activation and the kinases responsible for its phosphorylation in vivo have been unclear. The purpose of this study was to identify novel IRF5 kinases as potential targets for specific control of IRF5-driven inflammatory conditions. Based on a kinase inhibitor library screen and rounds of validation PYK2 and MEKK3 have been prioritised as candidate IRF5 kinases. Using the CRISPR-Cas9 system I have generated PYK2 and MEKK3 deficient macrophages. Deficiency in PYK2 had an effect on IRF5 activation, IRF5 recruitment to the genomic loci and transcription of IRF5 target inflammatory genes. Moreover, I have identified the PYK2 dependent tyrosine site within IRF5 and demonstrated its importance in IRF5 activation. PYK2 inhibition led to a comparable impact on macrophage transcriptomic signature as IRF5 deficiency, and suppressed cytokine production in biopsies taken from the inflamed gut mucosal tissue of patients with ulcerative colitis. The molecular dissection of the PYK2/IRF5 axis carried out in this thesis has a direct translational potential for inflammatory conditions such as ulcerative colitis, where both the PYK2 and IRF5 gene loci have been associated with increased genetic risk. Deficiency in MEKK3 had an effect on IRF5 activity and inflammatory gene expression. In the absence of MEKK3 inhibitors, clinically validated kinase inhibitors with off-target MEKK3 potency, were used as initial leads to design MEKK3-specific inhibitors. Chemical modifications of the lead compound Neratinib resulted in the identification and synthesis of analogs with comparable biochemical potency on MEKK3, improved selectivity on a small panel of pathway relevant kinases, with reduced IRF5-driven TNF activation in an IRF5- and MEKK3- specific manner.

Macrophages are multifaceted innate immune cells, able to adapt their phenotype to respond to a myriad of conditions, engaging in tissue-specific functions and mediating either inflammatory or anti-inflammatory responses depending on the encountered stimuli. They conduct key roles in the orchestration of immune responses; from pathogen recognition through sterilising inflammation to resolution and repair. The Udalova laboratory has previously demonstrated that IRF5 promotes a pro- inflammatory macrophage phenotype, leading to the secretion of TNF, IL-12, and IL-23, enhancing Th1/Th17-mediated immune responses, and described the cooperation between IRF5 and the transcription factor RelA, which mediate the production of pro-inflammatory genes. The aim of this thesis is to further characterise the activity of IRF5 in macrophage inflammatory responses. I demonstrate that IRF5 not only regulates the transcription of cytokines and chemokines in response to bacterial stimuli, but also anti-microbial peptides, whilst simultaneously down-regulating homeostatic and resolving macrophage functions. My data also suggests that IRF5 plays a role in enforcing monocyte to macrophage differentiation by up-regulating the transcription of key macrophages markers and repressing dendritic cell identity genes. To further characterise the mechanisms of the inflammatory response mounted by macrophages I used an unbiased approach; combining twenty-three transcription factor ChIP-seq data sets with chromatin accessibility information from ATAC-seq, uncovering RUNX1 as a novel partner of IRF5 that binds co-operatively to clusters of enhancers, which control the transcription of pro-inflammatory genes in a signal-dependent manner. This is the first study demonstrating a critical role for RUNX1 in activity of inflammatory macrophages.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6Chi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6Chi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b+ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

Changes to the composition and function of the gut mononuclear phagocyte (MNP) compartment are associated with the development of intestinal inflammation. Much work has focused on the role of MNPs in gut-associated lymphoid tissue in maintaining homeostasis, however little is known regarding the roles of MNPs during colitis. We have investigated MNPs in the large intestinal lamina propria during the steady state and inflammation. One of our primary aims was to determine the contribution of MNP subsets to intestinal pathology. For our studies of inflammation, we focused mainly on the Helicobacter hepaticus infection + anti-IL-10R model, which induces inflammation of the colon and caecum (typhlocolitis). We defined the composition of the MNP compartment alongside intestinal pathology scores throughout Hh + anti-IL-10R typhlocolitis. Peak pathology, 2-3 weeks after induction of colitis, coincided with peak frequencies of CX3CR1int Ly6C+ MNPs. Having observed the accumulation of CX3CR1int CD64+ monocyte/macrophage MNPs in the inflamed lamina propria, we conducted comparative whole genome microarray analysis of these cells isolated from the large intestine three weeks after Hh + anti-IL-10R treatment. CX3CR1int CD64+ MNPs selectively expressed a variety of pro- and anti-inflammatory genes, including a number of genes which individually can both promote and negatively regulate inflammation. IL-23 is essential for Hh + anti-IL-10R-induced intestinal pathology. We investigated the role of MNPs as a source of IL-23 which drives Hh + anti-IL-10R colitis. Unexpectedly, our results indicate that normally hyporesponsive CX3CR1hi macrophages may act as the initial source of IL-23, which induces development of colitis. Recruitment of Ly6C+ MHCII+ MNPs to the lamina propria was IL-23-dependent, and these cells also expressed IL-23, which may establish a positive feedback loop of immune cell recruitment, activation and IL-23 production. Finally, we also examined how MNPs might be recruited to the colonic lamina propria during inflammation. Our studies support the conclusion that CCR6 is not required for accumulation of monocyte-derived populations in the inflamed intestine. We cannot rule out a role for CCR2, however preliminary data from the Hh + anti-IL-10R colitis model suggest a potential role for CCR1 or its close relation CCRL2. Such pathways could represent new therapeutic targets in inflammatory bowel disease.

Macrophages are an integral part of the innate immune system and key players in pathogen clearance and tissue remodelling. Both functions are accomplished by a heterogeneous network of diverse macrophage populations that display pro- and anti-inflammatory properties. Understanding the molecular pathways that control this heterogeneity should provide abundant scope for the generation of more specific and effective therapeutics. Previously, our laboratory identified the transcription factor interferon regulatory factor 5 (IRF5) as the master regulator of the pro-inflammatory phenotype in human in vitro differentiated macrophages. Here, we show that murine macrophages with pro-inflammatory capacities in vitro and in vivo are also characterised by high levels of IRF5. IRF5 is upregulated in vitro in response to inflammatory stimuli such as GM-CSF and IFN-? and in a subset of in vivo macrophages in the arthritic knee joint during the model of antigen-induced arthritis (AIA). IRF5 is also expressed in other myeloid cells in vivo, especially in Ly6Chi monocytes and to a lesser extent in dendritic cells and neutrophils. We furthermore explored the role of IRF5 and IRF5-expressing macrophages in acute inflammation using two different models, AIA and acute lung injury, which are characterised by an extensive initial influx of neutrophils. Mice lacking IRF5 display reduced disease severity and decreased signs of inflammation in both models. Specifically, far fewer neutrophils accumulate at the site of inflammation. We investigated the molecular mechanisms underlying this phenomenon and discovered that IRF5-/- mice produced reduced levels of chemokines important for neutrophil recruitment, such as CXCL1. These studies extend our knowledge of the IRF5 expression pattern in vivo and suggest that in addition to the previously proposed role of IRF5 in chronic inflammation, it also has an important function in orchestrating initial acute responses. Thus, IRF5 blockade may be beneficial for both acute and chronic inflammation.