Macrophage-Regulatory T Cell Interactions Promote Type 2 Immune Homeostasis Through Resistin-Like Molecule α

RELMα is a small, secreted protein expressed by type 2 cytokine-activated “M2” macrophages in helminth infection and allergy. At steady state and in response to type 2 cytokines, RELMα is highly expressed by peritoneal macrophages, however, its function in the serosal cavity is unclear. In this study, we generated RELMα TdTomato (Td) reporter/knockout (RαTd) mice and investigated RELMα function in IL-4 complex (IL-4c)-induced peritoneal inflammation. We first validated the RELMαTd/Td transgenic mice and showed that IL-4c injection led to the significant expansion of large peritoneal macrophages that expressed Td but not RELMα protein, while RELMα+/+ mice expressed RELMα and not Td. Functionally, RELMαTd/Td mice had increased IL-4 induced peritoneal macrophage responses and splenomegaly compared to RELMα+/+ mice. Gene expression analysis indicated that RELMαTd/Td peritoneal macrophages were more proliferative and activated than RELMα+/+ macrophages, with increased genes associated with T cell responses, growth factor and cytokine signaling, but decreased genes associated with differentiation and maintenance of myeloid cells. We tested the hypothesis that RαTd/Td macrophages drive aberrant T cell activation using peritoneal macrophage and T cell co-culture. There were no differences in CD4+ T cell effector responses when co-cultured with RELMα+/+ or RELMαTd/Td macrophages, however, RELMαTd/Td macrophages were impaired in their ability to sustain proliferation of FoxP3+ regulatory T cells (Treg). Supportive of the in vitro results, immunofluorescent staining of the spleens revealed significantly decreased FoxP3+ cells in the RELMαTd/Td spleens compared to RELMα+/+ spleens. Taken together, these studies identify a new RELMα regulatory pathway whereby RELMα-expressing macrophages directly sustain Treg proliferation to limit type 2 inflammatory responses.

Pelvic pain correlates with peritoneal macrophage abundance not endometriosis

Endometriosis is a chronic neuroinflammatory pain condition affecting ~180 million women worldwide. Surgical removal or hormonal suppression of endometriosis lesions only relieves pain symptoms in some women and symptomatic relapse following treatment is common. Identifying factors that contribute to pain is key to developing new therapies. We collected peritoneal fluid samples and clinical data from a cohort of women receiving diagnostic laparoscopy for suspected endometriosis (n = 52). Peritoneal fluid immune cells were analysed by flow cytometry and data compared with pain scores determined using the pain domain of the Endometriosis Health Profile Questionnaire (EHP-30) in order to investigate the association between peritoneal immune cells and pain symptoms. Pain scores were not different between women with or without endometriosis, nor did they differ according to disease stage; consistent with a poor association between disease presentation and pain symptoms. However, linear regression and correlation analysis demonstrated that peritoneal macrophage abundance correlated with the severity of pelvic pain. CD14high peritoneal macrophages negatively correlated with pain scores whereas CD14low peritoneal macrophages were positively correlated, independent of diagnostic outcome at laparoscopy. Stratification by pain subtype, rather than endometriosis diagnosis, resulted in the most robust correlation between pain and macrophage adundance. Pain score strongly correlated with CD14high (P = 0.007) and CD14low (P = 0.008) macrophages in patients with non-menstrual pain and also in patients who reported dysmennorhea (CD14high P = 0.021, CD14low P = 0.019) or dysparunia (CD14high P = 0.027, CD14low P = 0.031). These results provide new insight into the association between peritoneal macrophages and pelvic pain which may aid the identification of future therapeutic targets. Lay summary Endometriosis is a common condition where cells similar to those that line the womb are found elsewhere in the body. It is associated with inflammation and pain in the pelvis and affects ~180 million women worldwide. Current treatments are not effective for all patients and we, therefore, need to understand what causes pain in order to develop new treatments. We investigated the types of immune cells present within the pelvis of women undergoing investigation for suspected endometriosis. Disease diagnosis and stage (I–IV) was recorded along with pain score determined by questionnaire. We characterised the immune cells present and compared them to disease stage and pain score. We found that pelvic pain was linked to the abundance of immune cells but, surprisingly, not to disease stage. These findings suggest that immune cells are closely associated with pain severity in endometriosis and may be good targets for future endometriosis treatments.

Embryonic-Derived Myb− Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb− “peritoneal-like” macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large “peritoneal-like” macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.

Macrophages inhibit and enhance endometriosis depending on their origin

Macrophages are intimately involved in the pathophysiology of endometriosis, a chronic inflammatory disorder characterized by the growth of endometrial-like tissue (lesions) outside the uterus. By combining genetic and pharmacological monocyte and macrophage depletion strategies we determined the ontogeny and function of macrophages in a mouse model of induced endometriosis. We demonstrate that lesion-resident macrophages are derived from eutopic endometrial tissue, infiltrating large peritoneal macrophages (LpM) and monocytes. Furthermore, we found endometriosis to trigger continuous recruitment of monocytes and expansion of CCR2+ LpM. Depletion of eutopic endometrial macrophages results in smaller endometriosis lesions, whereas constitutive inhibition of monocyte recruitment significantly reduces peritoneal macrophage populations and increases the number of lesions. Reprogramming the ontogeny of peritoneal macrophages such that embryo-derived LpM are replaced by monocyte-derived LpM decreases the number of lesions that develop. We propose a putative model whereby endometrial macrophages are “proendometriosis” while newly recruited monocyte-derived macrophages, possibly in LpM form, are “antiendometriosis.” These observations highlight the importance of monocyte-derived macrophages in limiting disease progression.

Recruited macrophages that colonise the post-inflammatory peritoneal niche convert into functionally divergent resident cells

Inflammation generally leads to substantial recruitment of monocyte-derived macrophages. What regulates the fate of these cells and to what extent they can assume the identity and function of resident macrophages remains unclear. We compared the normal fate of inflammation-elicited macrophages in the peritoneal cavity with their potential under non-inflamed conditions and in the absence of established resident macrophages. Following mild inflammation, elicited macrophages persisted for at least 5 months but failed to fully assume a GATA6hi resident identity due to the presence of enduring resident cells. In contrast, severe inflammation resulted in ablation of resident macrophages and a protracted phase wherein the cavity was incapable of sustaining a resident phenotype, yet ultimately elicited cells acquired a mature GATA6hi identity. Elicited macrophages also exhibited divergent features resulting from inflammation-driven alterations to the peritoneal cavity micro-environment and environment-independent features related to origin and time-of-residency. Critically, one environment-dependent feature of inflammation-elicited macrophages irrespective of severity of inflammation was a failure to produce the chemokine CXCL13, which correlated with a progressive loss in accumulation of peritoneal B1 cells post-inflammation. Hence, rather than being predetermined, the fate of inflammation-elicited peritoneal macrophages appears largely regulated by environment, changes in which result in long-term alteration in function of the peritoneal macrophage compartment post-inflammation.

Peritoneal macrophage phenotype correlates with pain scores in women with suspected endometriosis

ObjectiveTo characterise peritoneal macrophage populations in women with suspected endometriosis and assess if they are correlated with severity of pelvic pain symptoms.DesignFlow cytometry analysis of peritoneal fluid samples and clinical data.SettingUniversity Research Institute.PatientsClinical questionnaires, surgical data and peritoneal fluid were collected with informed consent from women undergoing diagnostic laparoscopy for suspected endometriosis (n=54).Intervention(s)NoneMain Outcome Measure(s)Severity of pelvic pain symptoms was assessed by the EHP-30 questionnaire. Immune cells recovered from peritoneal fluid were analysed by flow cytometry.ResultsPain scores (pain domain of EHP30) did not differ according to endometriosis diagnosis, stage of endometriosis or whether or not women were receiving hormone treatment. Analysis of immune cells in peritoneal fluid revealed two populations of peritoneal macrophages: CD14high and CD14low which were not altered by menstrual cycle phase or hormone treatment. CD14high peritoneal macrophages were increased in women with endometriosis compared to those without but were not altered by coincident reproductive health issues such as infertility or heavy menstrual bleeding. Peritoneal macrophage phenotype correlated with pelvic pain symptoms in women with suspected endometriosis. Notably, CD14high peritoneal macrophages negatively correlated with pain scores whereas CD14low peritoneal macrophages were positively correlated. This association was independent of endometriosis diagnosis.ConclusionPeritoneal macrophage phenotypes correlate with pelvic pain symptoms in women with suspected endometriosis and are altered by presence of disease. These results provide new insight into the association between endometriosis pathophysiology and pelvic pain symptoms.

Macrophages inhibit and enhance endometriosis depending on their origin

Macrophages are intimately involved in the pathophysiology of endometriosis, a chronic inflammatory disorder characterized by the growth of endometrial-like tissue (lesions) outside the uterus. By combining genetic and pharmacological monocyte and macrophage depletion strategies we determined the ontogeny and function of macrophages in a mouse model of induced endometriosis. We demonstrate that lesion-resident macrophages are derived from eutopic endometrial tissue, infiltrating large peritoneal macrophages (LpM) and monocytes. Furthermore, we found endometriosis to trigger continuous recruitment of monocytes and expansion of CCR2+ LpM. Depletion of eutopic endometrial macrophages results in smaller endometriosis lesions, whereas constitutive inhibition of monocyte recruitment significantly reduces peritoneal macrophage populations and increased the number of lesions. We propose a putative model whereby endometrial macrophages are pro-endometriosis whilst newly-recruited monocyte-derived macrophages, possibly in LpM form, are ‘anti-endometriosis’. These observations highlight the importance of monocyte-derived macrophages in limiting disease progression.