HMGB1, DNA, and HSPs 72 ; although a caveat to these findings was the possibility that the stimulatory activity of some of these putative autologous TLR ligands was actually due to contamination with microbial molecules and this was probably an artifact in some cases 73 , Nevertheless these findings raised the possibility that TLRs might be involved in sensing cell death, and in response, triggering inflammation.
Indeed some studies have found a potential role for TLRs in the inflammatory response to cell death. In these cases, DAMPs e. In addition, in a drug-induced liver injury model, a reduction in inflammation was observed in TLR9-deficient mice Overall the contribution of individual TLRs in the response to cell death, although varied, has been in general partial and in our hands relatively minor. A caveat to these loss-of-function studies is that if multiple TLRs participate in these responses so the loss of any one might have a minimal effect , then a larger role for these receptors could have been missed.
However one TIR adapter, myeloid differentiation factor 88 MyD88 , was essential for the inflammatory response to injected dead cells This was surprising, given the apparent limited role of TLRs in these responses, and suggested that other MyDdependent receptors might play a key role. Examination of mutant mice lacking such receptors revealed that the MyDdependent IL-1 receptor IL-1R was essential for the death-induced sterile inflammatory response In contrast, inflammatory responses to a microbial stimulus such as zymosan were intact in mice lacking the IL-1 pathway.
These data pointed to a key role of IL-1 in the death-induced sterile inflammatory response. Total neutrophil number in the peritoneal cavity was determined 14 h after stimulation. The finding that IL-1 played a dominant role in cell death-induced inflammation was surprising, because there are other proinflammatory cytokines produced in inflammatory responses that have redundant effects with IL Indeed, it may be generally true that IL-1 is a dominant cytokine driving sterile inflammatory responses in non-autoimmune situations see below.
Since IL-1 was required for the death-induced sterile inflammatory response, it was important to understand where it was coming from and how it was generated. One possibility was that the IL-1 was being released from the dying cells themselves This could occur because cells that are making IL-1 contain intracellular pools of the cytokine. Remarkably it is poorly understood how IL-1 is normally released from cells other than the fact that it does not follow a typical secretion pathway It has long been known that IL-1 can be released experimentally by breaking cells open, and therefore necrotic death, in which cells swell and lose integrity of their plasma membrane, is one way the cytokine could be released.
However, dendritic cells can make a lot of IL-1, and this IL-1 is sufficient to trigger inflammation even when live dendritic cells are injected in vivo Therefore, the IL-1 that is driving the sterile inflammatory response in many cases is not coming directly from the dead cell. In these situations, the IL-1 must be produced by cells in the host upon recognition of cell death. What is the host cell that is sensing cell death and in response producing the IL-1 that drives the sterile inflammatory response? The next question was which kind s of bone marrow-derived cell s was the source of IL-1 in these responses.
The fact that the cell was of bone marrow origin pointed to a leukocyte, but this did not narrow the field of candidates very far, as many of these cells can make IL To determine the identity of the relevant cell s , experiments were performed to assess the impact of eliminating various leukocytes.
The conditional depletion of cells expressing high levels of CD11b in CD11b-promoter-driven diphtheria toxin receptor transgenic mice markedly inhibited the inflammatory response to dead cells injected intraperitoneally Under these same conditions, the CD11b cell-depleted mice generated normal inflammatory responses to another proinflammatory stimuli injected intraperitoneally, such as MIP-2 Therefore, in the absence of the CD11b high cells, animals still have the necessary components to successfully mount inflammatory responses, but they are markedly impaired in doing so in response to cell death.
To determine whether this was the relevant cell type, macrophages were transferred back into CD11b-depleted mice. The adoptive transfer of these cells could reconstitute the response to dead cells 44 Fig. Even though highly purified macrophages supported the responses, a limitation of using primary cells is the possibility that a contaminating cell type was contributing to the reconstitution. To address this question, a cloned macrophage cell line was transferred, and it too was able to reconstitute responses Therefore, these studies indicated that macrophages are a key cell participating in the death-induced inflammatory response.
The role of dendritic cells in these responses was also investigated. This was of particular interest, because these cells do respond to dead cells 14 , 22 and a subset of them can express CD11b. However, conditional depletion of CD11c-expressing cells in a CD11c-promoter-driven-DTR transgenic mouse did not reduce the death-induced inflammatory responses in the peritoneal model, even though there was good depletion of dendritic cells Interestingly, adoptive transfer of dendritic cells into CD11b-depleted mice can also reconstitute the inflammatory to dead cells Fig.
Dendritic cells therefore have some capacity to participate in this response, and it is possible that in tissues where dendritic cells are more abundant than in the peritoneum, these cells might play a role in addition to macrophages. With the identification of a key role for macrophages in the death-induced sterile inflammatory response, the obvious question was whether these cells were the source of IL-1 that was needed for these responses.
To address this question, ILsufficient versus ILdeficient macrophages were transferred into CD11b-depleted mice. It was found that ILdeficient macrophages did not reconstitute inflammation, and they were indeed therefore a source of IL-1 in these responses As discussed above, urate is a proinflammatory DAMP that helps stimulate part of the inflammation to dying cells. Since this response is IL-1 dependent, it is perhaps not surprising that the inflammatory response to urate crystals was also found to require this cytokine 69 , In addition to urate crystals, there is a diverse set of particles that can induce inflammation in vivo.
These irritant particles include silica crystals, calcium pyrophosphate crystals, alum precipitated aluminum salt , and asbestos particles. The finding that inflammation to urate was ILdependent led to examination of the role of this cytokine in inflammatory responses to these stimuli and remarkably responses to all these particles were dependent on IL-1 25 , 80 - Subsequent studies found that particles that were not traditionally thought of inducers of inflammation, such as cholesterol crystals, would similarly stimulate ILdependent inflammation when they are injected or form in vivo 85 see below.
Inflammasomes consist not only of caspase 1 but also of a scaffolding protein called ASC and an NLR protein, the latter of which is thought to be the activating component of the complex Subsequently, it was found that NLRP3 was also required for macrophages to produce IL-1 when stimulated by many irritant particles, including silica, asbestos, alum, calcium pyrophosphate, and cholesterol crystals 25 , 80 , 82 , 83 , The ILdependent inflammatory response to cell death in vivo is significantly reduced in NLRP3-deficient mice 59 , 63 , Lai and Rock, unpublished result.
What is surprising and not understood is that the while priming is essential for responses to particles in vitro , the same particles stimulate ILdependent responses in vivo without a co-injected priming stimulus. This finding implies that something is providing the priming stimulus in vivo.
Conceivably the particles are stimulating the production of cytokine in vivo that prime macrophages. Indeed, it is clear that particles like urate crystals can stimulate cells through other pathways than the NLRP3 inflammasome 90 - Alternatively, it is possible that some of the ILproducing cells in vivo reside in a state where priming is not required, either due to their differentiation state or signals in the host environment e. In fact, in some of the examples discussed above where TLRs contribute to ILdependent sterile inflammatory responses, it is conceivable that they are doing so by helping to prime macrophages.
The NLR polypeptides are leucine-rich repeat proteins and are thought to function as intracellular sensors that control the activation of inflammasomes. In support of this concept mutations in NLRP3 lead to spontaneously active inflammasomes or ones that have a lower threshold of activation and as a consequence cause ILdependent autoinflammatory diseases Exactly what NLRP3 senses and how it activates the inflammasome is still not well understood.
A number of microbial molecules can stimulate the activation of NLRP3 inflammasomes and have been considered as putative ligands for this NLR protein 93 , although direct binding has not yet been documented. Since NLRP3 is a cytosolic and nuclear protein, it was a mystery as to how it could be sensing dead cells or irritant particles that are extracellular and excluded from the cell interior by the plasma membrane. When macrophages encounter these particles, they rapidly ingest them by phagocytosis, and this process was found to be essential for particle triggering of NLRP3-dependent IL-1 production As the next step in the pathway, there is evidence for two distinct mechanisms by which particles in phagosomes may stimulate NLRP3 Fig.
There are two distinct mechanisms proposed as to how NLRP3 inflammasomes are activated by sterile particles. The second model also requires phagocytosis of the sterile particles. In this pathway phagosomes acidify and the drop in pH causes Cathepsin Cat activation. Through some unknown mechanism, some of the particulate-containing phagosomes rupture and release their contents into the cytosol.
This vacuolar rupture is somehow sensed by NLRP3, possibly by binding a cleavage product of activated cathepsins or by the cathepsins cleaving NLRP3 in a way that activates it. In one pathway, internalized particles stimulate the production of reactive oxygen species ROS , and this process has been linked to the NLRP3 pathway because elimination of ROS with chemical scavengers sometimes inhibits the production of IL-1 Phagosomes contain an NADPH-oxidase complex that can be triggered by internalized particles to produce large amounts of ROS, which normally functions as part of the intracellular anti-microbial response.
This oxidative burst was initially thought to be the source of the reactive products driving the IL-1 response However, the particle-stimulated IL-1 response was subsequently found to be intact in cells that genetically lack NADPH oxidase, so this enzyme cannot be the only source of ROS that triggers this response 80 , 94 - More recently, it has been suggested that the ROS stimulating the NLRP3 inflammasome is instead coming from mitochondria 96 , although it is not yet clear how the ingestion of particles stimulates these organelles to make these reactive species.
In contrast to the above results, some other experiments have not observed any inhibition of particle-stimulated IL-1 responses by ROS scavengers and SOD1 mutations that lead to increased ROS levels decreased caspase 1 activation and IL-1 production 96 , 98 - While the reasons for these differences are not clear, they might be due to specific cell types or conditions used in different experimental systems.
It also suggests that there are additional pathways that can operate, and indeed there is evidence that this is the case. A second pathway also initiates when particles are internalized into phagosomes and starts with events that are part of normal phagosomal physiology Fig. As part of the process that coverts the phagosome into a catabolic vacuole, it fuses with lysosomes and a proton antiporter in the membrane acidifies the vesicle This drop in pH causes the lysosomal acid optimal proteases Cathepsins to become catalytically active, a process that is important for hydrolyzing the proteins that have been ingested by the phagocyte.
In addition, it turns out the activation of at least two of these proteases, Cathepsin B and L, also contributes to the triggering of the NLRP3 inflammasome, as discussed below. What happens next is that for reasons that are not yet understood, a fraction of the particle-containing phagosome rupture and release their contents into the cytosol While this event places the particles into the same subcellular compartment as the NLRP3 inflammasome, the particles themselves are not directly triggering this pathway.
Instead, it is the event of phagosomal rupture and specifically the release of Cathepsin B and L or the cleavage products from their substrates that seems to be somehow be sensed by NLRP3. In support of this concept, simply rupturing vesicles by osmotic lysis without particles activates the NLRP3 inflammasome in macrophages, and this process requires activated Cathepsins While most work has focused on how DAMPs stimulate cytokine production and inflammation, there may also be regulatory mechanisms that serve to limit these responses.
Thus, there are emerging data that at the same time that DAMPs stimulate inflammation, they also engage some regulatory receptors e. The finding that NLRP3 senses vesicular rupture has led to a new concept that this innate sensor is monitoring the health of cells and responds to internal cell damage.
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This may not be the only thing that NLRP3 senses, as there is evidence that it is stimulated by other molecules including products of microbes and ATP. However, at least some of these molecules are ones that can disrupt membranes e. NLRP3 is not the only example where the immune system is monitoring the health of cells.
Natural killer NK cell receptors monitor cells for MIC proteins, which are expressed under certain conditions of cell stress , It has been suggested that the release of heat shock proteins many of which are induced upon cell stress may stimulate innate cells , ; the release of HMGB1 upon cell damage can stimulate TLRs and RAGE 56 , Thus, an emerging theme is that beyond surveillance for infections the innate immune system may employ sensors to monitor the health of host cells and respond in ways to repair tissues or eliminate abnormal cells.
Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1α
Macrophages lacking any of the inflammasome components NLRP3, ASC, or caspase 1 make essentially no mature IL-1 when stimulated in culture with sterile particles 80 , However, the situation is more complicated in vivo. We and others have found that a variable but sometimes substantial sterile inflammatory response can be seen in caspase 1-deficient mice 69 , - Thus far we have focused on how IL-1 is produced. The next question is once IL-1 is produced, where is it acting to cause inflammation?
This has been investigated by examining where the IL-1R needs to be expressed. This receptor is broadly expressed on both leukocytes and tissue parenchymal elements , However, when IL-1R-deficient bone marrow is transplanted into wildtype mice, the chimeric animals mount perfectly normal sterile inflammatory responses, at least to crystals or dead cells 46 , This indicates that leukocytes or other bone marrow-derived elements do not need to be stimulated by IL-1 to generate the sterile inflammatory responses.
This was somewhat surprising, because IL-1 can induce more IL-1 It might have been expected that this autoamplification would have been important for optimal production of IL-1 from leukocytes, and this obviously cannot occur in the absence of the IL-1R. In contrast, if IL-1R-sufficient bone marrow is transferred into irradiated IL-1R-deficient mice, then the inflammatory responses are markedly attenuated.
These results indicate that the key target of IL-1 is a radio-resistant parenchymal element. The identity of this element s is presently under investigation. The host pays a price for mobilizing an inflammatory response.
It causes symptoms such as fever, malaise, and pain, which by themselves can compromise normal function. These products, while useful in the killing of microbes, can and do inflict damage in the tissue. Moreover, the bioactive mediators that are produced may act on tissue elements in ways that can lead to pathological changes e. However, in situations where the stimulus is sterile, the cost-benefit ratio may be less favorable.
The host response may contribute relatively little to defense, and the net effect may be damage. The host can deal quite well with small amounts of damage. There are a number of diseases that are caused by sterile inflammation and several of these are thought to be directly related to the NLRP3-IL-1 pathways described above.
Among these are a collection of diseases that are caused when particles, such as the ones discussed above, are deposited in tissues. When urate crystals form in the joints of hyperuricemic patients, the ILdependent inflammatory response causes the arthritis the disease of gout 46 , 79 , A similar problem occurs when crystals of calcium pyrophosphate deposit in joints, and when this happens, the particles cause the condition of pseudogout 79 , Another set of examples is when individuals inhale silica crystals or certain forms of asbestos that cause an inflammatory response in the lung that ultimately leads to pulmonary fibrosis in the diseases of silicosis and asbestosis Given these findings, it is possible that this response to inhaled particles may similarly contribute diseases caused too many other inhaled particulates such as those in cigarette smoke or air pollution.
The pathology caused by the innate response to particles may also contribute to a number of other conditions that are not traditionally thought of as particle-based diseases. One intriguing possibility is the disease of atherosclerosis.
The Regulation of Inflammation by Innate and Adaptive Lymphocytes
In this condition, plaques form in the walls of arteries when lipids and cholesterol deposit and the underlying smooth muscle cells proliferate When this process advances to a point where it interferes with blood flow then it causes ischemic disease. It is clear that what is driving this disease is a sterile inflammatory response in the vessel wall However, what is causing the sterile inflammation is unclear, other than it is related somehow to modified low density lipoproteins.
While it has long been recognized that cholesterol crystals are present in advanced atherosclerotic lesions, it has more recently been shown that microscopic cholesterol crystals can be found inside of macrophages as early as plaques can be detected Moreover, when such crystals are ingested or form in macrophages, they trigger the NLRP-3 inflammasome-dependent production of IL-1 in a cathepsin-dependent pathway Remarkably, blocking this inflammatory pathway in vivo reduces the inflammatory response to cholesterol crystals and markedly reduces plaque formation in experimental models of atherosclerosis.
There is also emerging data that IL-1 and the NLRP3 may be involved in metabolic syndrome in which obese individuals develop among other things insulin resistance and diabetes 6 , , At its core metabolic syndrome may be a sterile inflammatory disease of adipose tissue. What incites this inflammation is unclear, but in this condition, there is death of adipose tissue with subsequent phagocytosis of the fat cells by macrophages - There are also diseases that can be exacerbated by sterile inflammation. An example of this is disease processes that result in cell death. In this situation the inflammatory response to dying cells can compound and extend the tissue damage.
This is thought to occur e. In fact, it is likely that this process may to contribute to the pathogenesis of a number of diseases wherein pathological processes lead to cell death. In response to cell death, the innate immune system alerts the adaptive immune system to a potential problem in ways that promote the generation of responses. In parallel, it also rapidly mobilizes innate defenses to the site of injury through the generation of an inflammatory response.
Similar sterile inflammatory responses are generated to other kinds of non-microbial macroscopic particles. One of the important insights into these seemingly unrelated processes is that there is a common underlying pathway in which IL-1 is produced and is one of the key cytokines driving the ensuing inflammation. There are increasing insights into the identity of the cells that detect the sterile stimuli, how they sense such stimuli at the molecular level, and then respond to make IL This is important, because there is also increasing recognition that these processes contribute to the pathogenesis of a large number of diseases.
The authors thank Dipti Karmarkar and Hiroshi Kataoka for critical reading of the manuscript. There are no other potential conflicts of interest.
- Sterile inflammation of endothelial cell-derived apoptotic bodies is mediated by interleukin-1α.
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- Innate and adaptive immune responses to cell death.
- The Regulation of Inflammation by Innate and Adaptive Lymphocytes?
National Center for Biotechnology Information , U. MP, including endothelial MP 12 , are known to carry the surface proteins from their parent cells and to exert various proinflammatory properties as shown in rheumatoid arthritis The reasons for these discrepancies may be due to the difference of cell sources, activation mechanisms, and MP preparation methods, which remain major issues in this field 9. In addition, these particles are also different from MP because their size is larger than 0.
The characteristics of these particles are thus consistent with AptB 7 , 9. During apoptosis, the entire precursor translocates into the nucleus and remains tightly associated with chromatin The noninflammatory properties of apoptotic cells constitute a frequently accepted paradigm that when cell apoptosis occurs during the physiological processes of tissue senescence and renewal in the absence of any stress signal, there is little or no inflammation However, when cell apoptosis occurs in the context of an inflammatory stress, apoptotic cells form and release plasma-membrane—derived structures including MP and AptB.
These can be characterized by the presence of different surface markers, but these plasma-membrane—derived structures can possibly contain death-associated molecules that can amplify an inflammatory response Moreover, it has been shown that necrosis is also a programmed cell-death mechanism that can coexist with or follow apoptosis Interestingly, calpain is known to be activated during necrosis usually through calcium influx into the cell, but also under the action of caspase 3, a main player of apoptosis, through inactivation of calpastatin, an endogenous calpain inhibitor The equilibrium between cell death and macrophage phagocytosis is of major importance for homeostasis and perturbations of this equilibrium may lead to detrimental inflammation or auto-immune diseases 2 , For example, during severe tissue ischemia such as heart attack or stroke, in which massive cell apoptosis largely overcomes the phagocytosis ability of surrounding macrophages, secondary necrosis can occur and induce a pronounced neutrophilic inflammation.
In this situation, central nervous system neurons have indeed been shown to express active forms of both caspase 3 and calpain enzymes, thus coexistence of apoptosis and necrosis Alternatively, some individuals may have a defective clearance of AptB, as shown in systemic lupus erythematosus explaining why AptB persist in tissues, favoring the immunization against the nuclear components constitutive of the nucleosome structures 18 , Moreover, in these patients, proapoptotic and inflammatory situations such as UVB-exposure, a well-known lupus flare condition, may induce accumulation of secondary necrotic AptB and induction of inflammation.
Consistent with this hypothesis, the presence of circulating nucleosomes containing an active form of HMGB-1 has been recently reported in patients with systemic lupus erythematosus and found to favor a proinflammatory phenotype and anti-DNA immunization Fifteen-nanometer beads gold-labeled Ab was obtained from BB International. Protocol setting and gates were adjusted by using 0. Membranes were then incubated with respective secondary antibodies conjugated to horseradish peroxidase and specific bands revealed by using ECL Plus WB detection system.
Grids were negatively stained with 0. Animal studies were approved by the local ethical committee. Fifteen hours after the injections, animals were killed and peritoneal cavities were washed with 2 mL of PBS. Peritoneal fluid was harvested in EDTA tubes, and blood was collected by intracardiac puncture for serum collection. The authors declare no conflict of interest. This article contains supporting information online at www.
National Center for Biotechnology Information , U. Published online Dec 5. Contributed by Charles A. Dinarello, October 24, sent for review June 22, This article has been cited by other articles in PMC. Abstract Sterile inflammation resulting from cell death is due to the release of cell contents normally inactive and sequestered within the cell; fragments of cell membranes from dying cells also contribute to sterile inflammation.
Open in a separate window. Discussion At least two different particulate populations could be isolated from the supernatants of inflammatory apoptotic HUVEC. Materials and Methods Reagents and Antibodies.
Innate and adaptive immune responses to cell death
Generation, Numeration, and Characterization of Particulate Populations. Supplementary Material Supporting Information: Click here to view. Footnotes The authors declare no conflict of interest. The sterile inflammatory response. Decoding cell death signals in inflammation and immunity. Rider P, et al. IL-1alpha and IL-1beta recruit different myeloid cells and promote different stages of sterile inflammation. Immunological and inflammatory functions of the interleukin-1 family. Membrane interleukin 1 induction on human endothelial cells and dermal fibroblasts. Kaplanski G, et al. Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism.
Membrane vesicles as conveyors of immune responses. Proteomic analysis of dendritic cell-derived exosomes: A secreted subcellular compartment distinct from apoptotic vesicles. Microparticles as mediators of cellular cross-talk in inflammatory disease. Exposure to and engulfment of pathogen-derived products at the site of vaccination or infection activates APCs and triggers their production of inflammatory cytokines.
Both tissue-resident dendritic cells and macrophages display migratory behavior upon activation [ 29 , — ]. Interestingly, following infection with respiratory viruses such as influenza A virus, one APC subset, alveolar macrophages, becomes undetectable in the infected lung tissue until recruited monocytes are able reestablish the population [ ].
It remains unclear, however, whether the inability to detect alveolar macrophages following influenza is the result of their complete egress out of the tissue, a switch in their surface marker phenotype in response to the inflammatory milieu, or because of their elimination by the viral infection [ 29 , ].
The inflammatory mediators that these highly activated APCs produce and the surface costimulatory molecules that they express play a key role in shaping the ensuing adaptive immune response [ , ]. Vaccine strategies that specifically target pathogen-derived antigens to APCs in vivo [ ], that employ antigen-loaded dendritic cells themselves as the vaccine vehicles [ ], or that additionally trigger specific PRR receptors to direct T cell polarization are actively being explored as means to amplify the generation of effective T cell responses [ 8 — 10 ].
Such strategies are of particular interest for vaccination regimes for the elderly and cancer patients where the generation of effective immunity is challenging because of their compromised or suppressed immune states [ , ]. Under normal circumstances, the majority of expanded effector cells that migrate to sites of infection or antigen administration undergo contraction following subsequent pathogen or antigen clearance.
A small cohort of the expanded effectors will, however, survive to memory [ ]. Some antigen-specific memory T cells possess the ability to migrate throughout the body and are readily detected within tissues [ , ]. One subset of memory T cells, the tissue-resident memory T cell subset that does not circulate, is found exclusively within the tissues and may be strategically poised and specialized to perform sentinel functions [ — ]. Targeting the generation of tissue-resident memory T cells, especially for pathogens that infect mucosal tissues, is thus an attractive means to improve the efficacy of vaccines against pathogens that are not effectively controlled by traditional antibody-based approaches.
In addition to rapidly producing cytokines upon recognition of cognate antigen, memory T cells perform many other effector functions to protect the host against infection [ ]. These functions are, for the most part, recalled independently of most costimulatory molecules [ ]. For CD4 T cells, the best-known effector role is the provision of help for antigen-specific B [ ] and cytotoxic CD8 T cell responses as reviewed elsewhere [ , , ].
A novel effector role of memory T cells that is becoming more appreciated is the regulation of innate immune responses at sites of infection [ ]. Of importance to this discussion, memory T cells mediate rapid production of effector cytokines akin to the responses elicited from innate immune cells upon cognate encounter with specific pathogen-derived antigen. Memory T cells thus have the potential to act as powerful antigen-specific sentinels that are able to initiate rapid inflammatory responses against pathogens [ , — ].
Both memory CD4 [ , ] and CD8 [ , , ] T cells have the capacity to regulate and enhance the generation of early innate inflammatory responses within tissues upon cognate recognition of antigen. The antigen-specific regulation of inflammatory responses provides an additional means by which the immune response can generate alarm signals during infections with pathogens that possess means to evade detection by the innate immune-sensing mechanisms discussed earlier [ ]. It also provides a means whereby experienced memory cells can modulate the effector functions of the ensuing adaptive response of expanded secondary effector T cells that arise from resting memory T cell precursors during recall [ ].
Memory CD4 T cell-regulated enhanced inflammatory responses can also be initiated in the absence of infection. Indeed, the intranasal administration of cognate peptide antigen in the absence of any adjuvants or the administration of endotoxin-free protein that contains the epitope for which the cells are specific leads to the generation of potent early innate inflammatory responses [ ]. That memory cells do not depend on signal 2 to perform sentinel functions within the lung is in fitting with the observation that the activation and early recall of memory CD4 T cells in vivo are not affected by blockade of the CD28 costimulatory pathway [ ].
Similarities and inherent differences in the priming and function of memory CD4 and CD8 T cell responses are additional factors that must be considered in the design of innovative vaccination strategies that target the generation of protective antigen-specific T cells. Following secondary IAV infection, the earlier and more robust inflammatory response induced by memory CD4 T cells correlates with improved control of the virus in the lung [ ]. Innovative vaccines thus not only should target the induction of large numbers of memory T cells but also should strive to generate cells that possess optimal functional potential.
Current research employing high-dimensional mass cytometry that simultaneously measures over 40 parameters, including cell surface markers and intracellular proteins, as well as RNA expression at single-cell resolution [ ], will further advance our understanding of strong correlates of protection in specific models of infection. Such correlates will, in turn, help facilitate the development of optimal vaccination strategies. For the majority of innate immune cells, such imprinting results in a generic and nonspecific heightened inflammatory response that increases host antimicrobial defenses upon secondary infection.
Responses by NK cells may be an exception to this as they have been shown to display some elements of antigen-dependent memory [ — ]. It should be noted, however, that trained innate immune responses are functionally distinct from the highly specific recall responses characteristic of adaptive immune memory mediated by specialized subsets of CD4 and CD8 T cells and of antibody-producing B cells.
It has been long been appreciated that organs such as the lung remain in an altered state for an extended period of time following infection or insult [ , ]. The heightened inflammatory state that exists following the resolution of pathogen infection lasts for days, weeks, or even months and can provide a degree of nonspecific protection to unrelated pathogens.
Examples of heightened protective immunity induced by infection or vaccination are many and are discussed in detail elsewhere [ , , ]. A prime example is the ability of BCG vaccination, in mice as well as in humans, to increase resistance against a number of different pathogens [ — ]. Priming of innate immune cells resulting in increased nonspecific pathogen protection can also be caused by viral pathogens [ , ] and even exposure to pathogen-derived molecular patterns [ — ].
How conditioning of innate immune cells by the microbiota and infectious pathogens in human tissues influences the ability to generate protective immune responses following vaccination remains to be determined. Pathogen-associated encounters may not be the only events capable of training innate immune cells.
The engulfment of apoptotic host cells in the absence of infection has traditionally been considered an immunologically neutral event that fails to generate DAMP signals [ 33 ]. Recent observations, however, show that even this steady-state process can imprint macrophages for heightened inflammatory responses that mediate nonspecific resistance to microbial infection [ ]. These and other findings in a murine model [ ] suggest that most if not all tissue-resident macrophages become experienced during development by normal cellular turnover processes that educate them for future pathogen encounter.
The altered inflammatory state that exists following the resolution of infection can also have alternative and undesired outcomes. For example, conditioning of innate immune cells by prior infection can result in increased susceptibility to secondary infection [ ].
Increased susceptibility to secondary bacterial infection occurs following many respiratory virus infections [ ] and contributes markedly to the morbidity and mortality of disease [ ]. Mechanisms underlying increased susceptibility to secondary infection are many and include deficiencies in bacterial scavenging receptors such as MARCO on macrophages [ ], as well as the depletion of tissue-resident APC populations during primary infection [ ].
Increased production of inflammatory dampening cytokines IL and TGF-beta [ , ] and attenuation of protective host defenses through diminished production of IL-1b [ ], IL [ ], and antimicrobial peptides [ ] can also contribute to increased susceptibility. Increased expression of inflammatory dampening receptors such as CDR [ 66 , ] and differences in the chemotaxis, survival, phagocytic, and respiratory burst functions of neutrophils [ — ] may also lead to an inability of the innate immune system to contain and control secondary microbial threats following respiratory viral infection.
In addition to regulating early inflammatory responses that facilitate pathogen control, vaccine-induced T cell immunity may also be able to prevent these deficiencies in innate immunity as experimental evidence suggests that susceptibility to secondary bacterial infections is mitigated in primed animals in models of IAV infection [ ]. While highly specific in nature, the adaptive immune response can also alter the outcome of infections with seemingly unrelated pathogens.
This phenomenon, which has been termed heterologous immunity [ ], is mediated by cross-reactive T cells with T cell receptors that have the potential to recognize more than one peptide-MHC complex. For instance, in animal models of lymphocytic choriomeningitis virus LCMV , cytomegalovirus CMV , or IAV infection, prior virus-specific immunity has a beneficial impact on the outcome of subsequent vaccinia virus infection and results in improved viral clearance [ ]. Preexisting, heterologous immunity has been shown to alter protective T cell immunodominance hierarchies induced by primary infection.
It is argued that the presence of cross-reactive T cells narrows the virus-specific T cell repertoire and drives the selection of viruses able to escape adaptive immunity.