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Contents lists available at ScienceDirect

Immunology Letters

j ourna l ho me page: www.elsev ier .com/ locate / immlet

eview

he innate immune response

eo Koendermana,∗, Wim Buurmanb, Mohamed R. Dahac

Department of Respiratory Medicine, University Medical Center Utrecht, The NetherlandsSchool for Mental Health and Neuroscience, Maastricht University Medical Center, The NetherlandsDepartment of Nephrology, Leiden University Medical Center, The Netherlands

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a b s t r a c t

The innate immune response is of prime importance in the immediate recognition and elimination ofinvading micro-organisms. However, deregulation of this system is clearly associated with the patho-genesis of a wide range of inflammatory diseases. Innate immunity consists of a humoral and a cellular

eywords:nnate immune responseellular and humoral brancheuronal regulationranulocytesomplement system

branch, which are closely interacting. An additional level of control is found at the level of neuronalreflexes that can fine-tune these immunological mechanisms.

© 2014 Elsevier B.V. All rights reserved.

acrophages

. Introduction: the innate immune response: anvolutionary old defense mechanism

The human immune system consists of two branches: thentigen-specific adaptive immune response and the innatemmune response recognizing microbial associated molecular pat-erns (MAMP’s). The adaptive immune response is relatively slow,s antigen specific and requires gene rearrangement. The moleculesnd the receptors of the innate immune response on the other handre fixed present in the germ line DNA. Therefore, this system isuick as the system does not have to “learn”/adapt to a changingnvironment. This article will discuss a few important features ofhe innate immune system.

The development of the innate immune system predates thedaptive immune system by 500–700 million years [1]. Whereashe first indications of an antigen dependent adaptive responsessociated with T-lymphocytes are found in cyclostomata (e.g.amprey) that evolved around 300 million years ago [2], innatemmunity started already during the occurrence of multicellularrganisms 1 billion years ago [1]. A special form of phagocytosis,

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ndosymbiosis, is even much older. This latter process referso engulfment by single cells of targets, endo-symbionts thatould later evolve toward organelles such as mitochondria and

∗ Corresponding author at: Department of Respiratory Medicine, Heidelberglaan00, 3584CX Utrecht, The Netherlands. Tel.: +31 887557255.

E-mail addresses: L.Koenderman@umcutrecht.nl (L. Koenderman),.Buurman@maastrichtuniversity.nl (W. Buurman), M.R.Daha@lumc.nl

M.R. Daha).

ttp://dx.doi.org/10.1016/j.imlet.2014.10.010165-2478/© 2014 Elsevier B.V. All rights reserved.

chloroplasts [3]. The innate immune response is very complex anddifferent mechanisms evolved at different times during evolution[1].

1.1. Intracellular responses

One of the oldest immune responses that is found in bothplants and animals is RNA interference (RNAi) and other mech-anisms recognizing intracellular nucleic acids from pathogens[1]. Another mechanism is mediated by antimicrobial peptides.These mechanisms can be operational in both uni- and multi-cellular mechanisms and do not need any form of circulatorysystem.

1.2. (Para) cellular responses

When multicellular organisms developed, the immune systemadapted to this new situation by the evolution of immune mech-anisms that could respond extracellularly: the humoral responsemediated by single molecules and the cellular response medi-ated by effector cells. These two mechanisms have in commonthat a major part is mediated by proteins/receptors that rec-ognize microbe associated molecular patterns or MAMP’s. Thispattern recognition response allows the identification of patternsthat are present at multiple microbial products. A good example

l. The innate immune response. Immunol Lett (2014),

is the formyl-methionyl-leucyl-phenylalaline (fMLF) receptor thatrecognizes all proteins that start with the bacterial start sequenceformyl-methionyl. The innate immune response evolved into ahumoral part and a cellular part.

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.3. The Humoral innate immune response consists of two groupsf mechanisms: cascade systems such as the complement andoagulation systems and single antimicrobial molecules

The cascade systems are present in an inactive configurationre rapidly activated upon interaction with microbes. The singlentimicrobial proteins are either present in the intercellular spacer are produced by other cells or tissues during a stress or acutehase response [1]. The first indications of the evolution of theomplement system has been found in sea urchins which dates thevolution of this important cascade system (see below) to around50 million years ago [4].

.4. The cellular innate immune response

When organisms became more complex, part of the cells differ-ntiated into true immune cells. These cells acquired the propensityo eat or phagocytose targets such as bacteria. These phagocytesre found already in very simple multicellular organisms such asorals [5,6], which dates the evolution of these cells to 580 millionears ago. Hereafter, the innate immune cells started to evolve intoells with different functions: neutrophils, eosinophils, monocytes,acrophages.Starting 500 million years ago, a co-evolution of both innate and

daptive immune responses occurred, and a clear cross talk is nowresent in complex organisms such as vertebrates exemplified byhe evolution of dendritic cells and immunoglobulins [7].

. The humoral innate immune response

.1. The complement system (see Fig. 1)

As mentioned earlier, the humoral arm of innate immunity con-ists of a large number of players including chemokines, cytokines,efensins and complement. All these mediator systems provideefense at the initial phase of contact with pathogens and areesponsible to prevent potentially harmful infections. At the sameime the innate system presents antigens in an appropriate fash-on to innate immune cells, such as antigen presenting cells,

hich then subsequently induce the adaptive immune response.n this scenario the complement system is of major importanceecause it is involved in direct pathogen recognition and elimi-ation via opsonization and phagocytosis of potentially harmfulrganisms. Complement activation plays a major role in chemotaxisnd inflammation. Several functions of complement are essentialo enhance the potency of our innate immune defense by induc-ion of increased production of chemokines, cytokines and othernnate defense molecules. When complement is activated by aathogen, activated C3 namely C3b is deposited covalently on theseathogens. The deposition of C3b and its catabolic fragment C3desults in significantly increased recognition of antigens by follicu-ar dendritic cells and B cells and induction of the humoral adaptivemmune response [8] and production of antibodies and reactive Tells. In this regard it is known for a long time that for instanceats depleted of complement have a drastically reduced antibodyesponse against novel antigens [9]. Another very interesting func-ion of complement is the effect on clearance of soluble immuneomplexes and cell debris. The latter is of importance because itas been hypothesized [10] that large amounts of cell debris may

nduce an immune response against auto antigens and potentiallynduce autoimmunity.

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The complement system consists of a large number of com-lement components which are found mainly in the circulationut also in all tissues. Under physiological conditions the comple-ent system is activated to a very low degree but under conditions

activation of the terminal effector pathway and generation of the membrane attackcomplex C5b-9. The alternative pathway is dependent on interaction of hydrolyzedC3 and the alternative pathway components factor B, D and properdin.

of infection the system can be activated fully by three differentpathways. These three activation pathways of complement arereferred to as the classical, the lectin and the alternative pathway.Each of these pathways has its own soluble pattern like recogni-tion molecule(s). The classical pathway of complement is activatedmainly by antigen–antibody complexes (immune complexes), byseveral bacteria and their products, by apoptotic and necrotic tis-sue and cells. Here C1q is the recognition molecule leading tofurther activation of the classical pathway that involves C4, C2and generation of the classical C3 convertase C4b2a. A major rolefor the classical pathway is in the routing and elimination of sol-uble immune complexes. Following exposure to foreign antigensuch as found in food large amounts of antibodies are generatedand following re-exposure large amounts of immune complexesare formed in the circulation. These complexes can potentially bedeposited at random in small vessel walls and cause immune-complex-mediated inflammation. Several studies have shown thatimmune complexes in the circulation can activate the classicalpathway leading to deposition of C3b on their surface. C3b bearingimmune complexes are recognized by C3b receptors (CR1, CD35)on erythrocytes and subsequently transported to the liver wherethey are ingested and degraded. In this scenario the immune com-plexes are kept away from host endothelium and prevented fromdeposition in small vessels.

A major insight was recently gained on the role of the clas-sical pathway in the handling of apoptotic cells. Once apoptosisoccurs there is a flip-flop of the cell membrane, nuclear fragmen-tation and exposure of phosphatidyl serine. It was demonstratedthat apoptotic cells are major activators of the classical pathwayand that the degree of opsonisation by complement and comple-ment activation fragments. This potentially determines the rate ofelimination of cell debris and exposure of the adaptive immune sys-tem to autoantigens (3). The classical pathway is a major drivingforce in induction of inflammation in autoimmune diseases suchas Systemic Lupus Erythematosus (SLE). Next to complement acti-vation there is significant autoreactivity against host complementcomponents like C1q. These anti-C1q autoantibodies (C1qAb) wereshown to induce glomerular inflammation in mice [11] explain-ing earlier findings of a strong association between the presence ofC1qAb and renal involvement in patients with SLE [12].

The Lectin pathway is activated by complex carbohydrate stuc-tures and mediated via recognition molecules as Mannan binding

l. The innate immune response. Immunol Lett (2014),

lectin (MBL) and ficolins. This initial step leads to activation ofmannan associated serine protease-2 (MASP-2) and subsequentactivation of C4 and C2. This results in formation of the C3 conver-tase C4b2a, which is the same convertase generated via the classical

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athway. So, the lection pathway feeds into the classical pathwayt the level of C4 and C2. The lectin pathway is of major impor-ance for defense against pathogens especially when the adaptivemmune response is impaired such as in early childhood, advancedge or in patients under immunosuppression. In patients withrthotropic liver transplantation who are on immunosuppressiveherapy, major infections can occur especially in the first year afterransplantation. The liver is the major site of MBL production andnterestingly two major allotypes of MBL, of which one is the func-ionally active allotype exist. Therefore some patients receive a liverhat produces the active form while others receive the less activellotype of MBL; the patients receiving the livers with the activellotype of MBL are less prone to bacterial infections [13].

There are two sides to the same coin. MBL reacts with injuredissue or with dimeric IgA. In patients with primary IgA nephropa-hy, one of the major immune-mediated renal diseases in the world,he severity of renal disease is related to deposition of IgA in theidney. The driving force for inflammation is the activation of com-lement in the kidney. A seminal study demonstrated that bindingf MBL to IgA is an important biomarker for the severity of decline inenal function [14]. While MBL is protective against bacterial infec-ions in orthotopic liver transplantation the reverse is true for theecline of renal function after donor kidney transplantation. Herehe functionally active allotype of MBL was associated with moreevere decline in renal function in the transplanted graft [15]. MBLs on the one hand involved in complement activation but thereeems to be another function of MBL in for instance renal ischemianjury in rats. Here MBL is directly harmful to ischemic tubular cells16].

Evidence exists already for a long time that the C3 moleculendergoes continuous hydrolysis of an internal thioester hidden

n the molecule. Much light on this phenomenon of hydrolysisas provided by Law and Dodds [17]. Crystallization of human C3

y the group of Gros et al. from Utrecht University [18] has pro-ided detailed insight in the mechanisms of C3 activation and itsocking on pathogens and antigens [19]. Additionally the researchroup of Gros has contributed significantly to our insight in theechanisms of complement activation in general and especially in

he way several complement components interact with each otheruring activation of the complement system. Here extra insightas gained from escape mechanisms of Staphylococcus aureusave that prevent deposition of activated C3 on their surface viataphylococcal complement inhibitor (SCIN) elegantly shown byuzan Rooijakkers from the University Medical Center Utrecht [20].resently there are two potential ways of activation of the alterna-ive pathway namely one where hydrolyzed C3, C3(H2O) interactsith factors B and D to form the alternative pathway convertase3(H2O)Bb which then cleaves native C3 into C3b and formationf the amplification convertase C3bBb. This convertase is inher-ntly labile and under normal physiologic conditions is stabilizedy properdin [21].

More recent evidence has shown that several pathogens andpoptotic and necrotic cells can react directly with properdin thathen serves as a docking site for subsequent interaction with3(H2O) or C3b and generation of an alternative pathway C3 con-ertase [22,23].

To control the activation of complement several complementnhibitors are present that regulate the degree of complement acti-ation in the fluid phase and on host cells [18]. Especially the rolef factor H and factor I have received major attention. FH and FI aressential for the regulation and control of the amplification con-ertase and determine the turnover and activation of C3. When

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oo much C3 is activated for instance in several renal diseases, C3lomerolupathies [24], injury to small vessels and kidneys occur.ot only complement regulation is essential in these diseases butlso in diseases like age related Macula degeneration (AMD) [20].

PRESS Letters xxx (2014) xxx–xxx 3

Deficiencies and FI and FH are also associated with secondary C3deficiencies and susceptibility for several infections. FH is not onlyinvolved in regulation of complement activation in the fluid phase[25] but it provides additional protection against host complementof for instance endothelial cells against complement induced dis-eases like hemolytic uremic syndrome (HUS) [26].

Dysregulation of complement was shown to occur by autoan-tibodies against the C3Bb convertase itself. These autoantibodiesare known as C3 nephritic factors as identified by the group ofDaha from the Leiden University Medical Center (C3NeF). C3NeFstabilizes the C3Bb convertase and by that mechanisms gives theconvertase a much longer half live and a thereby a longer dura-tion of complement activation [27]. Thus hypercatabolism of C3 isa major driving force for a number of diseases where attack by hostcomplement is responsible for the induction of tissue inflamma-tion. Once tissue is injured, the tissue itself can serve as initiatorof complement activation and amplification of disease activity.Therefore it becomes more and more clear that implementationof complement control for the treatment of several diseases is ofmajor importance.

The generation of the amplification convertase C3bBb is stronglydependent on factor D which cleaves C3b-bound factor B into Bb.In patients with factor D deficiency, which were discovered for thefirst time at Leiden University Medical Center, strong susceptibilityfor bacterial infections is found [28,29]. These studies were fur-ther extended by the group of Professor Dirk Roos and Sanquinin Amsterdam who defined the molecular basis of the factor Ddeficiency in these patients.

2.2. Other soluble innate immune molecules recognizingmicroorganism associated molecular patterns (MAMPs)

During the acute phase response several proteins are found inthe peripheral blood that recognize MAMP’s. Many of these proteinsare relatively poorly defined in terms mechanisms and relativelyfew studies have been performed in The Netherlands. However,Haagsman et al. [30] from the university of Utrecht have shownthat surfactant proteins can play a role in innate immunity. Alsothe group of Prof. Hack [31] contributed significantly to the field byshowing that C-reactive protein (CRP), pentraxins and serum amy-loid P (SAP) are involved in the innate immune response. The groupof Prof. van der Poll and Herwald [32] has shown that the coagula-tion system plays in important role in the early immune response.Leemans et al. [33] showed that the calcium-binding protein com-plex S100A8/A9 plays an important role in the interphase betweeninnate immunity and tissue repair.

3. The cellular innate immune response

The cellular innate immune response is very complex and manycells are involved. These comprise of typical immune cells as well asnon-immunological cells. Together they form an important barrierto prevent pathogens to infect or affect the body.

3.1. Non myeloid innate immune cells

Many cells of the body contribute to the innate immuneresponse. These cells include epithelial cells, fibroblasts, etcthat basically form a barrier between the environment and themilieu interieur. Some of these cells make impenetrable materialsuch as cornified skin and shells or are involved in creating an

l. The innate immune response. Immunol Lett (2014),

anti-bacterial environment (e.g. low pH). During evolution manyof these cells acquired the potency to produce anti-microbialmolecules as well as immunomodulatory molecules such ascytokines and chemokines. These cells such as Paneth cells in the

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Fig. 2. Human myeloid innate immune cell. Cells were isolated from peripheralbw

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ut are an integral part of innate immunity but will not be theubject of this short review.

.2. Myeloid innate immune cells

The innate immune response is in part formed by cellsrom the myeloid lineage: monocytes, macrophages, neutrophils,osinophils, basophils, dendritic cells and platelets. All these cellsith specialized functions evolved from simple phagocytosing cellsuring evolution from simple multicellular life forms toward highlyomplex animals [3].

True phagocytes (see Fig. 2) are cells that have the capac-ty to recognize, engage, phagocytose and kill their targets by

ultiple cytotoxic mechanisms (see below). The true phagocyteompartment consists of monocytes, neutrophils, macrophagesnd eosinophils.

.2.1. MonocytesThese cells are traditionally seen as mononuclear phagocytes

hat are precursors for tissue dwelling macrophages. Monocytesriginate in the bone marrow from progenitor cells of the myeloidineage (CFU-GM) under control of different cytokines such as M-SF and activation of different transcription factors such as PU.1,

RF8 and KLF4 [34]. They are released to the blood where they areistributed before they home to different tissues. Here they dif-erentiate into macrophages specific for the tissue: e.g. Kuppferells in the liver and alveolar macrophages in the lung. Mono-ytes are relatively poorly phagocytosing cells when comparedith macrophages or neutrophils. These findings seem to imply

hat monocytes are a quite homogenous cell population of merelyacrophage progenitors. This simple view has been challenged

n recent years and many studies provide data for a much moreomplex situation. Now different types of monocytes are identifiedsee below) [35], and it has been shown that the cells can also berecursors of antigen presenting cells such as monocyte derivedendritic cells [36]. In addition, monocytes engage in immune reg-lation by producing large amounts of cytokines. The current view

s that monocytes are in fact very heterogeneous and are involved

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n immune regulation at different levels as well. Supportive forhis latter view is the large change in the monocyte populationuring chronic and acute inflammation [37]. This change is noweing developed as diagnostic tool in acute inflammation because

PRESS Letters xxx (2014) xxx–xxx

it is associated with inflammatory complications in trauma patients[37,38].

3.2.2. NeutrophilsIn marked contrast to monocytes, neutrophils are the profes-

sional phagocytosing cells present in both blood and tissues [39].The cells are primarily focused on targets that are smaller than theneutrophil itself and are eliminated inside the phagolysosome afterphagocytosis. The killing machinery (see below) is directed towardthe phagolysosome and under homeostatic conditions there is nocollateral damage by “spilling” these cytotoxic mediators into thepericellular space [39]. There is a clear debate as to how the con-trol of the neutrophil compartment is accomplished. The text booksdescribe a fairly simple view. Neutrophils are produced in the bonemarrow from the colony forming unit granulocyte/monocyte (CFU-GM) just as monocytes. However, G-CSF is the driving cytokine inproduction of neutrophils. The cells proliferate and differentiateuntil the metamyelocyte stage. Hereafter, the cells stop divid-ing and differentiate into cells first with a banded nucleus andthereafter with a segmented nucleus. This non-dividing phase isgenerally referred to as post-mitotic pool (PMT) and takes around5–6 days under homeostatic conditions [40]. Hereafter, the cellsleave the bone marrow under control of yet not completely iden-tified mechanism (s). However, both G-CSF and CXCR4 play animportant role. The cells are thought to circulate only for a shorttime (7–9 h) before going into the tissue [41]. In the tissue neu-trophils engage in immune surveillance by “looking for” targetsto eliminate. After a certain time that is yet to be determinedthe cells apoptoseis and are cleared by phagocytosis by reticu-lar macrophages. Recently, several studies have challenged thisview: (1) the half-life of neutrophils in peripheral blood underhomeostatic conditions seems much longer (2–3 days [42]), (2)neutrophil phenotypes have been described with different func-tions (see below [43]), (3) reverse migration of neutrophils fromtissue back to blood and lymph has been identified in both humans[44] and zebra fish [45], (4) neutrophils can transdifferentiate intoantigen presenting cells that can present antigens to the adaptiveimmune system [46], (5) neutrophils can produce a large array ofcytokines [47] and (6) an important part of neutrophils move backto bone marrow in a CXCR4 dependent fashion [48]. These find-ings indicate that neutrophils are both potent phagocytes as wellas immune-regulatory cells.

3.2.3. EosinophilsTraditionally eosinophils are thought to belong to a homogenous

cell population of cells that can engage with targets several timeslarger than themselves. For years investigators in the Netherlandshave worked on this interesting cell type in vitro: Yazdanbakhshet al. [49], Roos et al. [50], Koenderman et al. [51], Kaufman et al.[52], and Nijkamp et al. [53].

Eosinophils are involved in the defense against helminths [54].This immune response is in part mediated by TH2 cells and ismost likely the cause that eosinophils are involved in the patho-genesis of allergic diseases that are characterized by skewed TH2immunity. Not much detailed information is present regarding thecontrol of eosinophils in bone marrow, blood and tissue. The cellsoriginate from a colony forming unit (CFU)-eo/baso under the con-trol of IL-5 and GATA-1 and C/EBP� [55]. Again a PMT time ofaround 4 days precedes release of the cells to the peripheral blood.Detailed studies on the life spans of eosinophils in blood and tis-sue under homeostatic conditions are missing. However, the lifespan in peripheral tissue can be very long. Flood-Page et al. [56]

l. The innate immune response. Immunol Lett (2014),

showed in an elegant study applying a clinical anti-IL5 antibodyin asthma patients, that eosinophils can dwell in the bronchialtissue up to months under conditions that eosinophils in bonemarrow and blood are attenuated. This is in line with old studies

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y Rothenberg and colleagues [57] showing long term survival ofosinophils in vitro in eosinophil fibroblasts co-cultures. The fate ofissue eosinophils is uncertain. Recently, an elegant book discusseddditional functions of eosinophils in health and disease [54].

.2.4. MacrophagesMacrophages mainly originate from monocytes that migrated

rom the blood to the different tissues. It is still uncertainhether homogenous monocytes differentiate into different tis-

ue macrophages responding to local signals or that specializedonocytes specifically home to certain tissue sites. At the site,acrophages display multiple functionalities ranging from phago-

ytosis and killing of targets up to immune suppression by immuneuppressive macrophages [58]. Elegant work from The Netherlandsy Beelen and colleagues [59] as wells as Kraal et al. [60] has

dentified a clear functional polarization between pro – and anti-nflammatory macrophages.

Non-phagocytosing myeloid immune cells are basophils, den-ritic cells and platelets. These cells facilitate phagocytes toecognize and kill their targets. In addition, they play an importantole in immune regulation.

.2.5. BasophilsThe description of the function of basophils in immunity is

ainly connected to their role in allergic diseases. Their role inmmune homeostasis is fairly unknown, but seems similar to func-ions ascribed to mast cells. It seems that their potency to affectascular cells such that they facilitate transmigration of othermmune cells underlines their important role immune homeostasiss well as in tumor immunology [61]. The cells again are producedn the bone marrow from CFU-eo/baso under the control of IL-3.he PMT time is around 4 days and again little is known regardinghe half lives in blood and tissue. Importantly, basophils are non-hagocytosing cells and do not have the capacity to directly engageicroorganisms. In the Netherlands, pioneering work on the func-

ionality of these cells in vitro has been performed by Knol andlszewski [62].

.2.6. Dendritic cellsDendritic cells function at the interphase between innate and

daptive immunity. The cells randomly take up antigens, processhem and present the antigens to T-cells. They are from myeloidrigin and express multiple innate immune receptors. These impor-ant cells are beyond the scope of this small review, but theirunctions are excellently reviewed recently [63].

.2.7. PlateletsThese cellular fragments are important in coagulation. They

riginate by fragmentation from megakaryocytes that are cellsrom the myeloid lineage. Apart from their important role inoagulation these cell fragments contain a multitude of immuneegulatory proteins and other mediators. Their role in immune reg-lation is becoming more and more recognized. For a recent reviewee [64].

.2.8. Innate lymphoid cells (ILC)An exciting new concept has emerged on the interphase

etween adaptive and innate immunity: ILC’s. Recent studies per-ormed by Spitz and colleagues [65] have significantly contributed

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o the identification of these relatively rare lin-negative lympho-ytes not expressing a functional T-cell receptor. These cells playn important role in the immune responses particularly at barrierurfaces where they can quickly respond to immune triggers.

PRESS Letters xxx (2014) xxx–xxx 5

3.2.9. Innate immune receptorsA multitude of pioneering studies have been performed in The

Netherlands in the field of innate immune receptors. The groups ofRoos et al. [66], van de Winkel [67], Huizinga et al. [68], van Egmondand Bakema [69], Leusen et al. [70] and Koenderman et al. [71]have described the complex mechanisms underlying the outside-in and inside-out control of Fc-receptors in health and disease. Thegroups of van Kooyk and Geijtenbeek [72] have performed groundbreaking research in the area of C-type Lectins [72]. The groupsof L. Meyaard [73] and R. Hoek [74] described the importance ofimmune inhibitory receptors on innate immune cells. Van Strijpet al. [75] have identified important mechanisms by which com-plement receptors can bind to their ligand.

3.2.10. PrimingThe innate immune system faces an important dilemma. Fast

and robust cytotoxic responses are required to adequately engagewith pathogenic micro-organisms. However, aberrant or overzeal-ous activation of these responses can be deleterious to hosttissue when released into the pericellular environment. The innateimmune response has developed a mechanism that is generallyreferred to as priming to prevent hyperactivation of the system.Priming of phagocytes is defined as a mechanism by which a prim-ing agent can pre-activate and not activate a certain innate immuneresponse such that this response is amplified upon activation bya heterologous agonist [76]. Therefore, non-primed phagocytes inimmune homeostasis are characterized by a refractory phenotypethat is relatively difficult to activate by innate immune activatorssuch as formyl-peptides. However, upon interaction with immuneregulatory cytokines, chemokines and/or lipids, which by itself arepoor activators of cytotoxic mechanisms, the responsiveness ofphagocytes is greatly enhanced. Therefore, priming can be seen as asafe-lock in the prevention of tissue damage evoked by cytotoxicityof phagocytes. All phagocytes are controlled by priming at differ-ent levels as has been shown by Koenderman and colleagues as wellas the group headed by Roos: (1) adhesion and migration [77], (2)activation of cytotoxicity [78], (3) production of pro-inflammatorymediators [79], and interaction with opsonized particles [80]. Neteaand co-workers have shown that this priming behavior can persistfor days in monocytes and coined this persistent priming responseas trained immunity [81].

3.2.11. Phenotypes and functionalityAn exciting development in the field of phagocytes is the find-

ing that multiple functional phenotypes exist that are involvedin different parts of the innate immune response. Monocytes arenot homogeneous and several forms exist in the peripheral blood.The best illustration is characterization is the expression of Fc�RIII(CD16). Classical monocytes are CD16DIM whereas in recent yearstwo alternative phenotypes have been described characterized bya CD16BRIGHT phenotype [35]. The functional differences betweenthese monocytes are now being studied.

Recently, a population of myeloid cells has been identified withthe propensity that they can inhibit T-cell function. These cells,referred to as myeloid derived suppressor cells (MDSC’s), con-sist of young not well differentiated monocytes and neutrophils.They seem particularly important in tumor immunology, wherethey play a role in suppression of anti-tumor responses. This isbest studied in murine models [82]. Recently, MDSC’s have alsobeen found in humans. MDSC’s have been reviewed extensivelyin recent reviews [83]. The group of Koenderman and colleagues[43] identified another suppressive neutrophil phenotype has been

l. The innate immune response. Immunol Lett (2014),

described which occurs in peripheral blood during acute inflamma-tion evoked by LPS and/or trauma. This neutrophil is characterizedby a CD16BRIGHT/CD62LDIM phenotype and nuclear morphologyreminiscent for old cells (hypersegmented nucleus) [43]. It is not yet

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lear how many phenotypes are present in the neutrophil compart-ent and whether all phenotypes originate from one progenitor

ell or whether parallel pathways exist [83].Little to no clear data are present on functional phenotypes in

osinophils and basophils. The only old study showing “Th1” andTh2” type of eosinophils [84] has not been replicated by anothertudy.

It is clear that recent studies indicate that the phagocyteompartment in mammals is much more complex than ever antici-ated. Apart from being true phagocytes involved in direct killing oficrobes, the cells engage in a very complex network in regulation

f cross-talk between the two branches of the immune system

. Interplay of immune system and nervous system

The immune system and the nervous system are both impor-ant players in maintaining physiological homeostasis. Whereas themmune system and the nervous system have long been seen asutonomous systems, recent studies have provided evidence formport interactions between these systems. More over the ner-ous system can be regarded as an integrated regulator of immuneesponses. Watkins et al. [85] stated in their 1995 review that: “themmune system functions as a diffuse sense organ that signals therain about events occurring in the periphery”. The insight in the

nteractions between systems evolved quickly after the study oforovikova et al. [86] who reported that “Vagus nerve stimula-ion attenuates the systemic inflammatory response to endotoxin”.urther studies revealed that the nervous system has an activeodulating function in inflammation. The regulation of the magni-

ude of the inflammatory response is crucial: insufficient responsesan lead to infection and cancer, while excessive responses causeorbidity and mortality in autoimmune and infectious diseases.

t has now become more and more clear that this delicate bal-nce in response is not only regulated by the immune systemtself, but also fast and directly by the nervous system in additiono the hypothalamic-pituitary-adrenal axis. The so-called neuro-nflammatory reflex is able to quickly respond and regulate thenflammatory responses.

The work of Tracey’s group has led to much interest for theholinergic anti-inflammatory potential of the vagal system. Theynd others showed that electrical and pharmacological activation ofhe vagus inhibits many mostly innate immune responses rangingrom cytokine responses to bacterial toxins as endotoxin, inflam-

ation related to ileus, rheumatoid arthritis, colitis, hemorrhagichock, myocardial ischemia/reperfusion, experimental pancreatitisnd others [87–91]. The interaction of acetylcholine with nico-inic alpha7-ACh receptors (AChRs) on inflammatory cells plays anmportant role in the cholinergic anti-inflammatory pathway [86].his has led to studies using nicotine as anti-inflammatory agent inarious models.

Luyer et al. [92] showed in 2005 that also with nutritionalctivation of the vagus via CCK and CCK receptors significantnti-inflammatory effects were obtained. This nutritional pathwayppeared to reduce manipulation induced ileus, mast cell responsesnd endotoxin induced cytokine responses both in mice and inolunteers [93–98].

A non-pharmacological intervention could also be the use ofeuro-electronic implants that have shown remarkable therapeuticenefits for otherwise treatment-resistant disorders of the cen-ral nervous system [99]. This success has led to the developmentf vagal neurostimulation for the chronic inflammatory diseaseheumatoid arthritis [100]. Similar therapies are currently under-

Please cite this article in press as: Koenderman L, et ahttp://dx.doi.org/10.1016/j.imlet.2014.10.010

ay for application in postoperative ileus and inflammatory bowelisease, see ClinicalTrials.Gov.

It is only recently that interactions between sympathetic neu-ons, and their neurotransmitters, with the GI immune system

PRESS Letters xxx (2014) xxx–xxx

and bacterial flora have been appreciated. The postganglionicsympathetic neurons that supply the gastrointestinal tract havebeen visualized by the immunohistochemical localization of thenoradrenaline synthesizing enzymes, tyrosine hydroxylase (TH)or dopamine b-hydroxylase. A role of these sympathetic neu-rons seems relevant since vagus nerve terminals do not reach thegut mucosa directly, or synapse with gut immune cells directly,although direct interaction with the enteric neurons was shown[101]. In addition, vagal activation affects immune responses in thecolon [102] of which retrograde labeling experiments have shownthat only the proximal part is vagally innervated [101]. The roleof sympathetic neurons in the regulation of immune responsestherefore merits full attention in the near future.

Next to neuronal cells also other cells produce neurotransmit-ters as was already observed in 1929, by the neurophysiologist andNobel Prize winner Sir Henry Dale [103] who originally isolated thecholinergic neurotransmitter ACh from the spleen, a typical pri-mary lymphoid organ. Since a few years, we are aware of lymphoidcells producing ACh which were reported to be involved in thevagally activated inhibition of the response to endotoxin by splenicmacrophages [104]. More recent studies report that lymphocyte-derived ACh regulates local innate but not adaptive immunity [105].

An interesting target tissue could be intestinal epithelia. Cholin-ergic receptors are not only present on classical immune cells bothadaptive and innate but also on epithelial cells that play a role ininnate immunity such as enterocytes and Paneth cells [106–109].Paneth cells express a variety of cholinergic receptors includingmuscarinic and nicotinic alpha7-and beta 2 ACh receptors. Theseare functionally important because cholinergic agonists’ exposureto Paneth cells leads to massive antimicrobial peptide release. Inter-estingly no cholinergic synapses have ever been demonstrated onthese cells [110–113] suggesting the involvement of functionalalternative non-neuronal ACh producing cells. ACh producing lym-phoid cells are the principle candidates controlling the innateimmune function of these epithelial cells.

At this stage it has become clear that immune system and thenervous system are both important players in maintaining immunehomeostasis. To which extent the immune system plays a role inneuronal homeostasis is an interesting and most likely an impor-tant subject taking into account the multiple reports showing thatmental illnesses are associated with enhanced levels of inflamma-tory mediators.

Acknowledgements

Authors obtained funding of the Netherlands Organization forScientific Research (NWO) (Grant No. 053.21.112), the Dutch Kid-ney Foundation, the Lung Foundation Netherlands (Grant No.3.2.10.052), Dutch and German Cystic Fibrosis Foundations (GrantNo. COS21012), and The European Union (Grant No. 1209).

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