Researchers shown that monocytes are directly involved in the regulation of immune response

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Non-classical monocytes were long thought to play a purely surveillance role in the immune system. With the aid of a novel marker (PD-L1), Ludwig-Maximilians-Universitaet (LMU) researchers in Munich have now shown that they are directly involved in the regulation of immune response.

Monocytes comprise a functionally specific group of white blood cells and are known to play a role in the immune system.

Based on the sets of proteins found on their surfaces, they can be divided into two major subtypes, known respectively as classical and non-classical monocytes, which serve different functions in the immune system.

Up to now, non-classical monocytes have been viewed solely as surveillance cells that circulate in the bloodstream and serve to recruit other immune cells to sites of damage in the walls of blood vessels.

However, an international team led by Dr. Johan Duchêne, Mariaelvy Bianchini, Dr. Remco Megens and Professor Christian Weber of the Institute for Cardiovascular Prevention (IPEK) at the LMU Medical Center has now identified a specific marker for these cells.

Representative 3D reconstructions of end regions of bone showing classical monocytes (red spheres), monocytes in transition between conversion from classical monocytes to non-classical monocytes (pink spheres) and non-classical monocytes (violet spheres), as well vessels (green) in young and aged mice. Classical monocytes were significantly increased and NCMs were drastically reduced in aged mice. Credit: Bianchini et al., Sci. Immunol. 4, eaar3054 (2019)

Using this tool, they then went on to show, in a mouse experimental model, that non-classical monocytes also play a direct regulatory role in the adaptive immune response in certain tissues. The new study appears in the journal Science Immunology.

Classical monocytes migrate to sites of inflammation in the body, where they differentiate further to produce a range of specialized immune cells that activate other components of the immune response.

“We were interested in whether or not non-classical monocytes might also be able to regulate other types of immune cells in addition to acting as lookouts,” says Duchêne.

The problem was that no specific marker, with which they could be reliably identified, had been found for them. But this obstacle has now been overcome. In the course of their studies on mice, Duchêne and his colleagues characterized a specific surface protein as a suitable marker with which to track non-classical monocytes.

“The protein concerned (PD-L1) is a known and well-studied molecule that is found on the surface of cancer cells and serves to inactivate the immune response to malignant tumor cells. “It was a great surprise for us to discover that this protein is also strongly expressed on the surface of non-classical monocytes,” says Bianchini, who is first joint author on the study.

Video of two-photon laser scanning microscopy in blood circulation, revealing that PD-L1-expressing non-classical monocytes display characteristic patrolling behavior. Credit: Bianchini et al., Sci. Immunol. 4, eaar3054 (2019)

The new marker made it possible for the authors of the new study to show that classical monocytes, which develop in the bone marrow, are converted into non-classical monocytes when they first come into contact with specialized blood vessels in the vicinity of the cortical bone.

“This is the first experimental proof that both types of monocytes originate in the bone marrow.

Altered conditions in this microenvironment, caused by inflammation reactions, for instance can have a negative effect on the conversion process,” explains Megens.

“We have shown that this is in fact the case in the bone marrow of aging mice.”

Immunology -- not just supporting actors
A view from two-photon laser scanning microscopy showing a tertiary lymphoid organ (TLO) in the heart fat tissue enriched in clustered B cells (blue), T cells (green) and monocytes (red). Credit: Bianchini et al., Sci. Immunol. 4, eaar3054 (2019)

In addition, the team demonstrated that non-classical monocytes are not bit players in the adaptive immune system.

They can do more than acting as sentinels that raise the alarm.

They are able to infiltrate a specific type of inflamed tissue – known as tertiary lymphatic organs – for instance in the context of myocardial infarction, where they function as direct regulators of the adaptive immune response by modulating the activities of specific subsets of other immune cells.

“Our new marker has shown itself to be a very valuable tool, and it will help to further elucidate the biological roles of non-classical monocytes,” says Weber.

“It might even allow us to discover new molecular mechanisms that contribute to the development of inflammatory disorders such as cardiovascular disease and cancer.”


Monocytes and macrophages are members of the mononuclear phagocyte system, a component of innate immunity.

Monocytes are bone marrow derived leukocytes that circulate in the blood and spleen.

They are characterized by their ability to recognize “danger signals” via pattern recognition receptors.

Monocytes can phagocytose and present antigens, secrete chemokines, and proliferate in response to infection and injury.

Once recruited to tissues, monocytes are capable of differentiating into macrophages and dendritic cells.

Macrophages, on the other hand, are generally considered terminally differentiated cells that phagocytose pathogens or toxins, secrete chemokines to recruit other immune cells, and migrate to local lymph node beds via lymphatics where they present processed antigens.

Here we will discuss the current understanding of monocyte and macrophage function in lung immunobiology based on animal and human studies.

We will review their role in homeostasis and response to lung infection and tissue injury. Additionally, we will review their importance in lung transplantation with a focus on ischemia-reperfusion injury, primary graft dysfunction, and allograft rejection.

Monocytes and Macrophages in Lung Homeostasis

While once thought of as a lineage of cells with macrophages as the terminally differentiated cell in a progression from monocyte to macrophage, studies over the past twenty years have proven otherwise.

Advances in our understanding of monocyte and macrophage biology have direct applications to the lung and maintenance of its homeostasis. Together, in a coordinated fashion, monocytes and macrophages survey the lung (Figure 1A).

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Figure 1
Role of Lung Monocytes and Macrophages in Homeostasis, Bacterial Infection, Barotrauma, and Ischemia-Reperfusion Injury/PGD. (A) In homeostasis, lung non-classical monocytes patrol the endothelium, while classical monocytes survey the parenchyma. Classical monocytes can differentiate into interstitial macrophages or monocyte-derived dendritic cells, or remain undifferentiated while monitoring for infection or injury signals. Alveolar macrophages are resident in the lung, independent from monocytes and remain in the alveolar space. (B) During pathogen challenge in the alveolar space, alveolar macrophages are the first to be activated, releasing chemokines and cytokines which then recruit monocytes and neutrophils. Neutrophils, on arrival to the site of infection, are capable of degranulating and generating neutrophil extracellular traps in an effort to combat infection. (C) During mechanical ventilation, stretch-induced injury activates endothelium, alveolar macrophages, and epithelium, causing recruitment and activation of both classical and non-classical monocytes and the recruitment of neutrophils. (D) During ischemia-reperfusion, alveolar macrophages and monocytes become activated, recruiting neutrophils, and if the injury is severe enough, they can lead to primary graft dysfunction.

Monocytes

Much of our knowledge about monocytes comes from studies using mouse tissues and human blood. In both humans and mice, monocytes can be subdivided into: (1) classical inflammatory monocytes that are Ly6ChighCCR2+ in mice and CD14+CD16CCR2+ in humans; and (2) non-classical endothelial patrolling monocytes that are Ly6ClowCX3CR1high in mice and CD14dimCD16+CX3CR1high in humans (1). A third group of intermediate monocytes that are Ly6CintCX3CR1high in mice and CD14+CD16+CX3CR1high in humans have also been identified, but their specific role is incompletely characterized. It is likely that they have functions distinct from classical and non-classical monocytes (2).

Classical Monocytes

Classical monocytes are well-characterized.

In response to infection or injury, they proliferate in the bone marrow, are released into circulation in a CCR2-dependent manner and home to the site of interest via a chemokine gradient (3).

During bacterial infection, for example, these monocytes home to the site of infection and phagocytose pathogens, secrete a distinct set of chemokines that lead to recruitment of other immune cells, and present antigen via class II MHC (4).

Murine studies have also revealed that these monocytes can exit the vasculature, and without further differentiating, survey the tissue microenvironment, before departing via the lymphatics (5).

The spleen functions as a reservoir for monocytes. In response to signals emanating from a distant tissue injury, for instance release of angiotensin II during myocardial infarction, these cells can be mobilized to the site of injury from the spleen (6).

In the lung, these cells are capable of differentiating into pulmonary dendritic cells and macrophages. Utilizing a combination of fate-mapping techniques, parabionts, and fluorescent reporter murine studies, investigators have shown that Ly6Chigh classical monocytes differentiated into both the CD11b+CD103 and CD11bCD103+ dendritic cell subsets (7).

In addition, these cells differentiate into macrophages when recruited to the lung and help replenish the tissue resident macrophage pool when depleted either by injury or through experimental manipulation (8).

Non-classical and Intermediate Monocytes

Non-classical endothelial patrolling monocytes were first reported in 2007 (9) and studies of their biology and function remain ongoing.

These cells are likely the descendants of classical monocytes that have returned to the bone marrow and under the control of Nur77 (Nr4a1) mature into non-classical monocytes (10).

They display a distinct motility and crawling pattern. In vivo imaging studies have shown non-classical monocytes crawling along the luminal side of the endothelium. This integrin-dependent crawling is independent of the direction of blood flow.

In fact, it is frequently against the direction of flow (1112). In the lung, non-classical monocytes are known to be capable of differentiating into CD11b+CD103 dendritic cells (7).

Recent studies have further attempted to explore the role of these monocytes. Investigators have shown that non-classical monocytes are involved in intraluminal surveillance of the endothelium and phagocytosis of injured endothelium along with recruitment of neutrophils to the site of injury (13).

In addition, these cells have also been shown to limit tumor metastases to the lung via the CX3CR1-CX3CL1 axis (14). Studies of human non-classical monocytes have shown that these monocytes sense nucleic acids and viruses via TLR7 signaling and can initiate the innate immune response by secreting cytokines (15).

These monocytes can be mobilized from the marginalized vascular compartment during vigorous exercise, sepsis, and after cardiac surgery (1618) and are depleted by high-dose glucocorticoid treatment (1920).

However, there are conflicting reports regarding the function of non-classical monocytes.

Some studies suggest this population secretes pro-inflammatory cytokines, while others suggest they are anti-inflammatory and promote the resolution of inflammation and the initiation of healing and fibrosis (14).

Difficulty differentiating non-classical monocytes from intermediate monocytes by cell surface marker expression may account for the conflicting reports regarding the function of these cells.

In fact, as non-classical monocytes have become the subject of recent more intense investigation, it has become clear that there is a population of “intermediate” monocytes.

Murine Ly6C and human CD14 are expressed in monocytes at high, intermediate, and low levels as a continuum, and intermediate levels of these surface markers along with expression of the cell surface marker 6-sulfo LacNac (slan) identify the intermediate monocyte subset. When studied as distinct populations, intermediate monocytes show higher expression of MHCII and are more closely related to classical than non-classical monocytes based on transcriptome analyses (2122).


More information: M. Bianchini el al., PD-L1 expression on nonclassical monocytes reveals their origin and immunoregulatory function, Science Immunology (2019). immunology.sciencemag.org/look … 6/sciimmunol.aar3054

Journal information: Science Immunology
Provided by Ludwig Maximilian University of Munich

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