CXCR2 deficient mice display macrophage-dependent exaggerated acute inflammatory responses

CXCR2 is an essential regulator of neutrophil recruitment to inflamed and damaged sites and plays prominent roles in inflammatory pathologies and cancer. It has therefore been highlighted as an important therapeutic target. However the success of the therapeutic targeting of CXCR2 is threatened by our relative lack of knowledge of its precise in vivo mode of action. Here we demonstrate that CXCR2-deficient mice display a counterintuitive transient exaggerated inflammatory response to cutaneous and peritoneal inflammatory stimuli. In both situations, this is associated with reduced expression of cytokines associated with the resolution of the inflammatory response and an increase in macrophage accumulation at inflamed sites. Analysis using neutrophil depletion strategies indicates that this is a consequence of impaired recruitment of a non-neutrophilic CXCR2 positive leukocyte population. We suggest that these cells may be myeloid derived suppressor cells. Our data therefore reveal novel and previously unanticipated roles for CXCR2 in the orchestration of the inflammatory response

Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1β release through pyrophosphates

In acute inflammation, extracellular ATP activates P2X7 ion channel receptors (P2X7R) on M1 polarized macrophages to release pro-inflammatory IL-1β through activation of the caspase-1/nucleotide-binding domain and leucine-rich repeat receptor containing pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X7R on these macrophages nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL-1β remains high and the inflammasome is intact, P2X7R activation selectively uncouples to the NLRP3-inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts through its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1β release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments

Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex

The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome

Image based machine learning for identification of macrophage subsets

Macrophages play a crucial rule in orchestrating immune responses against pathogens and foreign materials. Macrophages have remarkable plasticity in response to environmental cues and are able to acquire a spectrum of activation status, best exemplified by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time consuming, require large numbers of cells and are resource intensive. In this study, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus we suggest high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers

Btk regulates macrophage polarization in response to lipopolysaccharide

Bacterial Lipopolysaccharide (LPS) is a strong inducer of inflammation and does so by inducing polarization of macrophages to the classic inflammatory M1 population. Given the role of Btk as a critical signal transducer downstream of TLR4, we investigated its role in M1/M2 induction. In Btk deficient (Btk (−\−)) mice we observed markedly reduced recruitment of M1 macrophages following intraperitoneal administration of LPS. Ex vivo analysis demonstrated an impaired ability of Btk(−/−) macrophages to polarize into M1 macrophages, instead showing enhanced induction of immunosuppressive M2-associated markers in response to M1 polarizing stimuli, a finding accompanied by reduced phosphorylation of STAT1 and enhanced STAT6 phosphorylation. In addition to STAT activation, M1 and M2 polarizing signals modulate the expression of inflammatory genes via differential activation of transcription factors and regulatory proteins, including NF-κB and SHIP1. In keeping with a critical role for Btk in macrophage polarization, we observed reduced levels of NF-κB p65 and Akt phosphorylation, as well as reduced induction of the M1 associated marker iNOS in Btk(−/−) macrophages in response to M1 polarizing stimuli. Additionally enhanced expression of SHIP1, a key negative regulator of macrophage polarisation, was observed in Btk(−/−) macrophages in response to M2 polarizing stimuli. Employing classic models of allergic M2 inflammation, treatment of Btk (−/−) mice with either Schistosoma mansoni eggs or chitin resulted in increased recruitment of M2 macrophages and induction of M2-associated genes. This demonstrates an enhanced M2 skew in the absence of Btk, thus promoting the development of allergic inflammation

Physiological roles of macrophages

Macrophages are present in mammals from midgestation, contributing to physiologic homeostasis throughout life. Macrophages arise from yolk sac and foetal liver progenitors during embryonic development in the mouse and persist in different organs as heterogeneous, self-renewing tissue-resident populations. Bone marrow-derived blood monocytes are recruited after birth to replenish tissue-resident populations and to meet further demands during inflammation, infection and metabolic perturbations. Macrophages of mixed origin and different locations vary in replication and turnover, but are all active in mRNA and protein synthesis, fulfilling organ-specific and systemic trophic functions, in addition to host defence. In this review we emphasise selected properties and non-immune functions of tissue macrophages which contribute to physiologic homeostasis

The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance

Macrophages in inflammatory multiple sclerosis lesions have an intermediate activation status

BACKGROUND: Macrophages play a dual role in multiple sclerosis (MS) pathology. They can exert neuroprotective and growth promoting effects but also contribute to tissue damage by production of inflammatory mediators. The effector function of macrophages is determined by the way they are activated. Stimulation of monocyte-derived macrophages in vitro with interferon-γ and lipopolysaccharide results in classically activated (CA/M1) macrophages, and activation with interleukin 4 induces alternatively activated (AA/M2) macrophages. METHODS: For this study, the expression of a panel of typical M1 and M2 markers on human monocyte derived M1 and M2 macrophages was analyzed using flow cytometry. This revealed that CD40 and mannose receptor (MR) were the most distinctive markers for human M1 and M2 macrophages, respectively. Using a panel of M1 and M2 markers we next examined the activation status of macrophages/microglia in MS lesions, normal appearing white matter and healthy control samples. RESULTS: Our data show that M1 markers, including CD40, CD86, CD64 and CD32 were abundantly expressed by microglia in normal appearing white matter and by activated microglia and macrophages throughout active demyelinating MS lesions. M2 markers, such as MR and CD163 were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. Double staining with anti-CD40 and anti-MR revealed that approximately 70% of the CD40-positive macrophages in MS lesions also expressed MR, indicating that the majority of infiltrating macrophages and activated microglial cells display an intermediate activation status. CONCLUSIONS: Our findings show that, although macrophages in active MS lesions predominantly display M1 characteristics, a major subset of macrophages have an intermediate activation status

Chikungunya Disease: Infection-Associated Markers from the Acute to the Chronic Phase of Arbovirus-Induced Arthralgia

At the end of 2005, an outbreak of fever associated with joint pain occurred in La Réunion. The causal agent, chikungunya virus (CHIKV), has been known for 50 years and could thus be readily identified. This arbovirus is present worldwide, particularly in India, but also in Europe, with new variants returning to Africa. In humans, it causes a disease characterized by a typical acute infection, sometimes followed by persistent arthralgia and myalgia lasting months or years. Investigations in the La Réunion cohort and studies in a macaque model of chikungunya implicated monocytes-macrophages in viral persistence. In this Review, we consider the relationship between CHIKV and the immune response and discuss predictive factors for chronic arthralgia and myalgia by providing an overview of current knowledge on chikungunya pathogenesis. Comparisons of data from animal models of the acute and chronic phases of infection, and data from clinical series, provide information about the mechanisms of CHIKV infection–associated inflammation, viral persistence in monocytes-macrophages, and their link to chronic signs

Identification and manipulation of tumor associated macrophages in human cancers

Evading immune destruction and tumor promoting inflammation are important hallmarks in the development of cancer. Macrophages are present in most human tumors and are often associated with bad prognosis. Tumor associated macrophages come in many functional flavors ranging from what is known as classically activated macrophages (M1) associated with acute inflammation and T-cell immunity to immune suppressive macrophages (M2) associated with the promotion of tumor growth. The role of these functionally different myeloid cells is extensively studied in mice tumor models but dissimilarities in markers and receptors make the direct translation to human cancer difficult. This review focuses on recent reports discriminating the type of infiltrating macrophages in human tumors and the environmental cues present that steer their differentiation. Finally, immunotherapeutic approaches to interfere in this process are discussed