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IL-10 Part 2: Viral Persistence In EBV, and HIV; Potential IL-10 Strategies For Viral Clearance (Programmed Death Ligand 1)

This article is not medical or healthcare advice. Before starting any health related regimen seek the advice of your Primary Care Physician or an M.D.


The Teaser - IL-10 Has Viral Homologs - That Enable Viral Persistence!

"The role of virus-encoded IL-10 homologs is likely to provide a tool to enable modulation of the local immune response so as to enhance the capacity to replicate, disseminate, and/or persist in an otherwise immunocompetent individual. In fact, there is emerging evidence that virus-encoded IL-10 homologs function in this capacity in a variety of settings."[1]


Viruses Modulate Immune Function Through IL-10

"An important role of the potent immunomodulatory cytokine IL-10 in virus infections has become increasingly clear in recent years, and the acquisition of IL-10-like sequences by viruses represents a remarkable evolutionary step which likely equips the virus with a powerful tool to modulate host immune function. A range of both human and nonhuman viruses have been shown to encode IL-10 homologs, and the number will undoubtedly grow as new sequence information becomes available. Whether in acute, persistent, or latent infection settings, the expression of virus-encoded homologs of IL-10 would serve as an efficient means to interfere with multiple immune components to ensure successful infection of the host. To date, all of the virus-encoded IL-10 homologs tested have demonstrated immunomodulatory properties, although these homologs do not always mimic the full range of properties encompassed by their cellular counterparts, nor by each other, highlighting the complexity of this means of virus-encoded immune control. While there is still much to be understood about the full extent of the direct and indirect impacts of vIL-10 on the host immune response to virus infections, their discovery and the functional studies performed so far provide a fascinating insight into the capacity of some viruses to acquire a potent weapon with the potential to significantly influence the course of infection."[1]


Uh Oh - EBV Has IL-10 Homologues.

"A tool that has proved to be valuable in studying IL-10 is the Epstein–Barr-Virus BRCF-1 gene product, which has been termed vIL-10 because of its significant sequence as well as structural homology. Although vIL-10 has a significantly reduced affinity for IL-10R , it shares many suppressive properties of hIL-10, including inhibition of IFN-γ production by activated human PBMCs, while stimulatory effects were only detected by hIL-10 treated cell lines."[2]


IL-10 Viral Homologues Limit The Capacity Of Host Immune Response To Clear Infection

"The capacity of viruses to successfully infect the immunocompetent host to cause disease argues in favor of virus-encoded functions that specifically target components of the immune system so as to orchestrate an environment that limits the capacity of the host immune response to clear infection. In this respect, many viruses have evolved to coexist with the host immune system by developing an arsenal of strategies to avoid immune surveillance and elimination from the host. These include viruses which have acquired homologs of cellular cytokines or cytokine receptors as a strategy to limit host immune recognition. Cellular interleukin-10 (IL-10) is a pleiotropic immunomodulatory cytokine produced by a wide variety of cells, including monocytes, macrophages, T and B lymphocytes, dendritic cells (DC), keratinocytes, epithelial cells, and mast cells........the key features of this cytokine (IL-10) relate mainly to its capacity to exert potent immunosuppressive functions on the expression of a range of cytokines and chemokines.... The immunosuppressive properties of IL-10 are primarily restricted to cells of the myeloid lineage . "[2]


IL-10 Induces JAK/Stat Pathway

"In contrast, IL-10 has been shown to exert a stimulatory effect on B lymphocytes , mast cells , thymocytes , and CD8+ T cells , highlighting the cell-type-dependent immunomodulatory properties of this cytokine. The immunomodulatory functions manifested by IL-10 require engagement of this cytokine with its cell surface bound receptor. The IL-10 receptor (IL-10R) consists of two different subunits (IL-10R1 and IL-10R2) . IL-10 binds first to IL-10R1 with high affinity, and the resulting intermediate IL-10/IL-10R1 complex then binds with lower affinity to IL-10R2. The resulting active signaling complex induces the JAK/Stat signal transduction pathway. "[1]


Viruses Up-Regulate IL-10 Enhance Infection Via Immuno Suppression

In the context of viral pathogenesis, infections with a number of different viruses have been documented to upregulate the expression of IL-10, and in some cases, this upregulation has been shown to enhance infection by suppressing the immune function, suggesting that the far-reaching effects of this cytokine have many advantages for invading pathogens."[1]...."....suggesting that EBV IL-10 (ebvIL-10) could act on multiple cell types and inhibit cytokine synthesis by both T cells and NK cells."[1]


Yes, Small Genetic Mutations Can Have Large Effects

"Since the viral gene was most likely captured from the host and evolved over time to retain properties that were most beneficial in promoting virus persistence, these differences in biologic activity were not completely unexpected. Substitution of an alanine at this position in hIL-10 resulted in loss of stimulatory activities, while immunosuppressive functions were not affected. The wild-type ebvIL-10 protein normally contains an alanine at this position, and when a mutation substituting isoleucine for the alanine was made, the viral cytokine gained the ability to stimulate proliferation of MC/9 cells, although the proliferation was not as robust as that observed with wild-type hIL-10. These results demonstrate that even a single amino acid can impact a cytokine's functional repertoire."[1]


IL-10 is a key player in the establishment and perpetuation of viral persistence

"The immune system has evolved multipronged responses that are critical to effectively defend the body from invading pathogens and to clear infection. However, the same weapons employed to eradicate infection can have caustic effects on normal bystander cells. Therefore, tight regulation is vital and the host must balance engendering correct and sufficient immune responses to pathogens while limiting errant and excessive immunopathology. To accomplish this task a complex network of positive and negative immune signals are delivered that in most instances successfully eliminate pathogen. However, in response to some viral infections, immune function is rapidly suppressed leading to viral persistence. Immune suppression is a critical obstacle to the control of many persistent virus infections such as HIV, hepatitis C and hepatitis B virus, which together affect more than 500 million individuals worldwide. Thus, the ability to therapeutically enhance immunity is a potentially powerful approach to resolve persistent infections. The host derived cytokine IL-10 is a key player in the establishment and perpetuation of viral persistence. this chapter discusses the role of IL-10 in viral persistence and explores the exciting prospect of therapeutically blocking IL-10 to increase antiviral immunity and vaccine efficacy."[3]


EBV, Re-Activation, ImmunoSuppression, CD8 T Cells Physically Deleted or Exhausted

"In most situations this concerted effort of innate and adaptive responses is effective in eliminating the pathogen. However, in some cases the acute resolution of infection is incomplete and viral persistence results. Herpes simplex virus, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV), along with γ2-herpes virus in mice, are hallmark examples of infections that develop lifelong viral persistence by ‘hiding’ from the immune response. This presence of latent/reactivating infection is associated with functional T cell responses that control viral replication upon re-emergence. In contrast, human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections in humans, and lymphocytic choriomeningitis virus (LCMV) infection in rodents establish persistent infections characterized by sustained high levels of viral replication and immunosuppression. In response to these persistent infections, virus-specific CD4 and CD8 T cells are physically deleted or persist in an attenuated (termed exhausted) developmental program unable to proliferate to viral antigens or produce important antiviral and immunostimulatory cytokines (e.g., IFNγ, TNFα, IL-2) . The physical deletion of high affinity CTL and the low amount of remaining virus-specific CD8 T cells, in conjunction with the loss of cytokine production, the inability to proliferate to viral antigen and attenuated CD4 Th cell and B cell responses all culminate in the failure to purge infection. The exhausted state is characterized by a unique transcriptional profile featuring up-regulation of the transcription factor Blimp1 and inhibitory cell surface molecules, such as programmed death receptor 1 (PD1), along with down modulation of cytokine and TCR signaling molecules. Thus, T cell exhaustion is a unique T cell developmental program, still active and exerting some control over virus replication, but distinct from the productive T cell responses during acute infection or the anergic/tolerant responses to self-proteins.

Although potentially counter-intuitive, induction of this exhausted state is an important mechanism"[3]


Host-based suppressive factors inhibit clearance of persistent viral infection (PD-L1)

"Both general and virus-specific immune suppression has been well established during persistent viral infection, however, the mechanisms that govern this phenomenon have only recently been brought to light. Many persisting viruses (e.g., HCMV, HIV, HCV) encode proteins that actively suppress immunity either by direct inhibition of T cell responses and/or by down-regulating antigen recognition molecules. Other persistent viruses (e.g., LCMV) do not encode suppressive factors yet their infection still rapidly leads to a suppressive state. This suggests that the inability to rapidly control infection triggers a suppressive program within the host.

The seminal discovery by Rafi Ahmed and colleagues that blockade of the host-encoded protein programmed death (PD) ligand 1 (PD-L1) restored function to exhausted CD8 T cells and enhanced control of persistent LCMV infection, led to the realization that the host itself potentiates immune suppression during viral persistence. The suppressive role of PD-1/PD-L1 interaction was promptly demonstrated to suppress CD8 T cell responses to a variety of diverse persistent infections in vitro including the RNA viruses HIV (a retrovirus) and HCV (a flavivirus) and the DNA virus, HBV (a hepadnavirus) as well as in vivo against SIV (a non-human primate retrovirus). The diversity of these viruses with respect to replication strategies, target organs and infected cell types highlights the conserved role of PD-1/PD-L1 mediated immunosuppression and establish that the host is a powerful inhibitor of T cell immunity during persistent infection."[3]


Multiple Factors Suppress T Cell Responses During Viral Persistence

Shortly following the identification of PD-1 mediated immunosuppresion during viral persistence, the dominant role of IL-10 in attenuating effector T cell responses to initiate persistent infection was established. Infection of mice with a persistent, but not an acutely cleared variant of LCMV leads to sustained expression of IL-10 by multiple immune cell subsets and functional exhaustion of CD4 and CD8 T cells. However, when IL-10 activity was neutralized, either using mice genetically deficient in IL-10 expression or antibodies that block the IL-10 receptor (IL-10R), immune function was sustained and the otherwise persistent virus was rapidly cleared. Persistent LCMV replicated to high titers in wild-type and IL-10 deficient mice 5 days post infection and prior to the onset of T cell responses. By day 9, T cells did not lose function in IL-10 deficient mice and they were able to clear persistent LCMV whereas viral titers remained high in wild type mice. CD8 T cells were required for this clearance since depletion of CD8 T cells in IL-10 deficient mice prior to infection led to LCMV persistence (E. Wilson and D. Brooks, unpublished observation). This was the first identification that a single factor was responsible for derailing the immune response to permit viral persistence and importantly, that sustaining T cell immunity could facilitate the clearance of an otherwise persistent infection.

Subsequently, multiple immunoregulatory factors were identified to limit T cell responses during viral persistence, including TGFβ, Tim3, CTLA4, CD27/CD70. Exhausted CD8 T cells simultaneously express multiple negative regulatory factors during persistent infection and these factors can simultaneously, but via different pathways, limit T cell activity. Further, a single suppressive factor can differentially affect distinct T cell populations. For example, blockade of CTLA4 during persistent LCMV infection did not impact CD8 T cell responses in vivo, whereas it did enhance HIV-specific CD4 T cell responses in vitro. Similarly, IL-10 directly limits CD4 T cell responses, but not CD8 T cell responses, to an acute LCMV infection. In addition to T cells, IL-10 and other suppressive factors modulate multiple immune subsets including B cells, DC, macrophages and NK cells to contribute to enhanced virus control. The diversity in suppressive mechanisms provides the potential opportunity to individually manipulate T cell responses (particularly in combination with therapeutic vaccines) to produce the optimal effector response required to control a specific viral infection. Antibody therapies that block multiple suppressive pathways additively increase antiviral T cell activity. Thus, while increased production of suppressive factors by the immune system itself ultimately leads to the demise and failure of antiviral immunity, excitingly these factors can be inhibited for therapeutic benefit."[3]


Impact of reversing T cell exhaustion during viral persistence

"Understanding the mechanisms and coordination of the multitude of suppressive factors involved in immunoregulation is crucial to the design of effective antiviral therapies. Therapeutic strategies that target host-based factors to restore immune function are less susceptible to resistance via viral mutation as they do not target a specific viral protein. Thus, blockade of host-based negative regulatory factors could be effective against diverse persistent viruses that induce T cell exhaustion without engendering viral resistance.

Although extremely promising, the efficacy of blocking suppressive factors to enhance antiviral immunity in humans remains unclear....... However, recent evidence blocking PD-1 in SIV infected rhesus macaques suggests that these blockade strategies may be effective . Short-term PD-1 blockade (4 treatments over 10 days) provided long-term restoration of T cell responses that correlated with enhanced SIV control. Interestingly, memory B cell responses and SIV-specific antibody production were also increased following PD-1 blockade. The reason for the increased B cell responses was not elucidated and could be due to either direct or indirect mechanisms (e.g., enhanced CD4 T cell help to B cells), but does indicate the exciting prospect that targeting a single molecule may simultaneously enhance multiple arms of the immune response culminating in virus control.

In total these studies indicate the incredible effect of blocking regulatory factors to enhance T cell function during viral persistence; however, this is not without potential negative impact. These dominant regulatory factors and pathways are instilled to prevent errant or unrestricted immune responses. Even in the absence of overt infection, deficiencies in these factors can lead to the massive expansion of effector-like T cells and a variety of autoimmune disorders. In response to an infection, the inability to attenuate T cell responses can lead to severe immunopathology and death. For example, persistent LCMV infection is fatal in PD-L1 knockout mice and while IL-10 deficient mice survive and clear persistent LCMV infection they are more susceptible to death in response to higher doses of persistent LCMV as compared to IL-10 sufficient hosts (D.G. Brooks, unpublished observations). Further, treatment with the immunostimulatory cytokine IL-21 during the early phase of persistent LCMV infection dramatically elevated virus-specific CD8 T cell responses and mortality. Thus, although detrimental to virus clearance, it is likely that the increased expression of negative regulatory factors and T cell exhaustion is a conserved and rapid mechanism to prevent lethal immunopathology when the host ‘senses’ that virus replication has out-competed the immune response to it. In some instances enhanced immunopathology is observed in the absence of IL-10 regulation without an effect on viral replication, as is the case during a neurotropic model of mouse hepatitis virus infection. On the other hand, once chronic infection has been established and T cell numbers have contracted, reversing exhaustion appears to be well handled in animal models of LCMV and SIV infection. The relationship between T cell exhaustion (i.e., attenuating T cell responses), excessive immunopathology and host survival must be carefully considered when optimizing therapies targeting host suppressive factors, particularly if instituted early during persistent virus infection.

Despite freeing virus-specific T cells to fight infection, blockade of regulatory factors may simultaneously unleash the regulation of self-specific immune cells or ‘tolerant’ immune cells in multiple organs and in the case of IL-10 blockade, particularly in the gut. Such a result could have the unintended consequence of triggering autoimmunity or immune responses to ingested food or endogenous enteric bacterial microbiota. It should be noted that overt autoimmunity was not observed in our studies when IL-10R blockade was implemented during the chronic phase of LCMV infection (D.G. Brooks, unpublished observation) nor following PD-1 blockade in SIV-infected macaques. However, these studies utilized short-term treatment regimens and longer term therapy or different individuals dependence on a particular pathway to maintain immune homeostasis could affect negative responses. Thus, while therapies that block negative regulators of immune function clearly hold tremendous antiviral potential the possible consequences should be carefully evaluated."[3]


Il-10 Role In Viral Persistence

"IL-10 was initially known as cytokine synthesis inhibitory factor (CSIF) and was first identified as a CD4 produced Th2 cytokine with the ability to indirectly repress Th1 responses. It is now evident that multiple cell types including DC, B cells, macrophages, CD4 T cells, CD8 T cells, NK cells as well as innate and adaptive regulatory T cells can produce IL-10.

The IL-10 receptor (IL-10R) is a class II cytokine family member composed of two subunits, IL-10R1 is the unique ligand binding subunit and IL-10R2 is the signaling subunit that is shared with other family member cytokines (IL-22, IL-26, IL-28 and IL-29). Dimerization of the receptor by IL-10 results in signaling through STAT3 and activation of gene expression. Specificity of IL-10 responsiveness is dictated both by the expression of IL-10R1 and availability of the cytokine. While the signaling subunit (IL-10R2) is constitutively expressed by most cells, IL-10R1 is differentially regulated by activation in a cell type specific manner in hematopoietic cells and is inducible on non-hematopoietic cells, illustrates some of important targets of IL-10 signaling in persistent viral infections.


IL-10 aborts T cell responses when present during priming and can inhibit ongoing T cell activity to viral infections. It acts directly on antigen presenting cells to decrease stimulatory molecule expression (i.e., MHC class I and class II, B7-1, B7-2), alter cytokine production and prevent maturation ultimately dampening T cell activation. In addition to these indirect effects, IL-10 can also act directly on T cells to limit proliferation, functional differentiation and effector activity. Although controversial, emerging data also indicates that genetic polymorphisms in the IL-10 promoter that result in lower IL-10 production are associated with clearance of HCV infection and enhanced virus control during chronic HCV, HBV, HIV and EBV infections further supporting the important role of this cytokine in host immunosuppression.

Counter to its negative regulatory functions, IL-10 can positively stimulate NK cells, in some instances CD8 T cells and induce B cell proliferation and antibody production . Thus, although generally immunosuppressive, IL-10 can function through a variety of mechanisms (likely simultaneously) to fine-tune the pathogen-specific immune response. In total, these data emphasize the diverse functions (many of which may be pathogen specific) that IL-10 plays to regulate multiple immune parameters.

IL-10 plays a major role in limiting autoimmune disease under steady-state conditions by controlling circulating, self- or gut microbiota-reactive T cells. Consistent with this role, IL-10 knockout (KO) mice develop inflammatory bowel disease (IBD)/colitis approximately 8 weeks after birth. Both CD4 T cells as well as resident enteric bacteria were required for the emergence of colitis indicating that under steady-state conditions IL-10 serves to suppress immune activation against the endogenous gut microbiota. Although autoimmunity was not generally observed in other organs of IL-10 deficient mice it is likely that the cytokine also negatively regulates immunity in other tissue compartments. Specific incidence of IBD and colitis demonstrate the key importance of IL-10 in immune homeostasis to gut antigens."[3]


IL-10 - Sources, Mechanisms, Targets During Viral Persistence - Its Virus Specific!!!!

"...IL-10 can be produced by a variety of cell types to regulate their own and other cells functions. During persistent virus infection multiple cell types have been identified to produce IL-10, generally with immunosuppressive effects. The dominant IL-10 producing cell type varies with different virus infections likely reflecting inherent differences in the pathogen-specific response as well as tissue-specific immune regulation. Although individual IL-10 expressing cells may exert some level of suppression, it is likely that during persistent virus infections the suppressive state mediated by IL-10 is maintained by multiple cells types that individually limit a variety of immune parameters, with the final effect being the inability to clear infection.

CD4 T cells were the first identified and are probably the most recognized IL-10 producing cell type. Upon activation and in response to the antigenic environment CD4 T cells differentiate into multiple Th subsets. These different subsets have both unique and overlapping qualities with multiple Th subsets capable of producing IL-10. Initially identified as a Th2 cytokine, IL-10 can also be produced by Th1, Th17, T follicular helper and both natural (Foxp3+) and induced (Foxp3−) T regulatory (Treg). In general, IL-10 production by CD4 T cells during viral persistence is associated with the inducible Tr1 Treg population consisting of both virus-specific and non-specific cells. The mechanism of Tr1 cell emergence in persistent infection is not known. They may arise non-specifically as a bystander effect of general T cell activation, they may be preferentially induced in an effort to limit excessive immunopathology or they could have deliberately evolved to arise in instances of prolonged immune activation. Finally, a variety of IL-10 expressing CD4 Th cells may be lumped into the Tr1 category with ‘regulation’ as only one of their potentially diverse functions.

The actual impact of Treg produced IL-10 in limiting virus-specific responses during persistent viral infection remains largely unclear. IL-10-mediated Treg activity during viral persistence is observed following infection of mice with Friend virus (FV). IL-10 expressing CD4 Treg cells are activated following FV infection and limit antiviral CD8 T cell responses in vivo facilitating increased persistent virus replication. However inhibition of Treg activity alone did not enhance control of virus replication, which required the adoptive transfer of large amounts of FV-specific CD8 T cells that could now function in the absence of IL-10 signals. Interestingly, FV-induced immunosuppression was IL-10 dependent, however IL-10 was not the Treg effector mechanism required to suppress CD8 T cells. IL-10 present during CD4 T cell priming can induce anergy, but also programs the development of additional IL-10 producing CD4 T cells. In such a manner, Treg produced IL-10 could promote the differentiation of more Treg cells as opposed to directly suppressing antiviral function, the latter being performed by other Treg produced inhibitory mechanims. Thus, IL-10 would be important for suppressing T cell responses, but would not itself be the direct effector mechanism.

Early following persistent LCMV infection, virus-specific IL-10 producing CD4 T cells are observed, but IL-10 protein expression rapidly decreases in conjunction with other Th1 cytokines. IL-10 producing CD4 T cells are observed throughout persistent LCMV infection; however, they are relegated to the non-virus-specific CD4 T cell subset and (D. Brooks, unpublished observation). Further, the amount of non-LCMV-specific IL-10 producing CD4 T cells in the spleen is similar during acute and persistent LCMV infection (E. Wilson, D. Brooks, unpublished observation). It will ultimately be interesting and important to determine whether these non-LCMV-specific CD4 T cells regulate the virus-specific immune response and the outcome of deleting these cells toward clearance of persistent LCMV infection.

In addition to CD4 T cells, IL-10 producing CD8 T cells, monocytes/macrophages, dendritic cells, B cells and NK cells are observed during persistent viral infections. During persistent LCMV infection IL-10 is produced by multiple cell types including NK cells, DC and B cells all of which likely contribute to immune regulation. MCMV persistence in the salivary gland of infected mice is dependent on IL-10 producing CD4 T cells, whereas B cell produced IL-10 suppresses CD8 T cells in the spleen during MCMV infection. Thus, different cell types utilize IL-10 in a compartmentalized fashion to suppress distinct facets of immunity in the same host during persistent infection. A multi-cell mediated IL-10 response is also observed during HIV infection, with monocyte/macrophages often comprising the largest IL-10 producing subset in the peripheral blood. However, whether IL-10 producing cells differ in PBMC and tissue during HIV infection, and if so, what cells produce IL-10 in various tissue compartments, remains to be determined.

It will ultimately be critical to establish whether persistent viral infections are the result of IL-10 production by a single cell type with other IL-10 producing cells playing an auxiliary role or whether IL-10 production by multiple cell types is necessary. In the case of the former it will be important to define what cell type produces the ‘relevant’ IL-10 and how it aborts immunity. In the latter case, how each cell type suppresses individual immune components will need to be determined. Answers to these questions are critical from both a biologic standpoint to define the pathogenesis of persistent infection as well as from a therapeutic standpoint to modulate IL-10 expression by cells inhibiting antiviral activity while leaving other IL-10 producing cells intact to prevent autoimmunity and immunopathology."[3]

Back To IL-10 Homologues

"Perhaps most strongly corroborating the importance of IL-10 toward viral persistence, several persistent viruses encode their own IL-10 homologs, including EBV, HCMV and some poxviruses to modulate the immune response and facilitate replication, spread and/or persistence. The first viral IL-10 (vIL-10) homolog to be identified was encoded by EBV with ebvIL-10 exhibiting ~70% amino acid sequence identity with human IL-10 (hIL-10). EBV infects B cells leading to latent infection and in some cases B cell transformation. Both hIL-10 and ebvIL-10 have similar immunosuppressive activity and stimulated B cell proliferation, differentiation and antibody production. However, ebvIL-10 had ~1000-fold lower affinity for the cellular IL-10R and failed to promote MHC class II upregulation by B cells or to inhibit IL-2 production by CD4 T cells. Thus, in addition to its suppressive role permitting immune escape, an important function of ebvIL-10 may be to target B cell proliferation and differentiation thereby increasing the amount, permissiveness and/or transformation of infected cells without affecting the immune-stimulatory capacity. Similarly, HCMV encodes an IL-10 homolg with 27% identity to hIL-10. hIL-10 and cmvIL-10 exhibit similar immunosuppressive and stimulatory characteristics: inhibiting LPS-induced DC maturation, cytokine production and upregulation of multiple T cell co-stimulatory molecules. cmvIL-10 also inhibited type I interferon production by pDC, a major source of type-I interferon during viral infection. As discussed, type I interferons stimulate the virus-specific immune response and trigger a general antiviral state, but they also potently block HCMV infection. As a result, cmvIL-10 may enhance the spread of HCMV while simultaneously suppressing the early immune response. Interestingly, in vivo infection of mouse DC by murine CMV (MCMV) induced many of these same immunosuppressive effects despite not encoding an IL-10 homolog. Thus, in addition to the direct affect of HCMV encoded IL-10, HCMV replication in DC in vivo may itself trigger an immunosuppressive program. During viral latency HCMV produces a shorter differentially spliced IL-10 variant sharing some of the immunosuppressive qualities of the cmvIL-10 produced during productive infection (e.g., down-regulation of MHC class II on monocytes) however, this homolog did not suppress DC maturation, co-stimulatory molecule induction or induce proliferation of a B cell line. The decreased expression of MHC II inhibited CD4 T cell identification of latently infected cells allowing HCMV to evade immune recognition without affecting other immune functions that may compromise infection. Thus, in its lifecycle, HCMV utilizes different IL-10 mediated suppressive mechanisms at different stages to persist."[3]


Elevated IL-10 and Viral Replication

"One constant among persistent viruses is elevated expression of IL-10 and its direct correlation with virus replication. In addition to stimulatory factors, virus replication inherently triggers counter-regulatory measures to ultimately contain the immune response. Many signals inherent to immune activation induce IL-10 expression, but the precise ‘sensors’ of prolonged/heightened virus replication during persistent infection have yet to be determined. Pathogen specific IL-10 induction is likely achieved through the integration of multiple virus- and host-derived mechanisms and therefore will likely be dictated in a conserved and in a pathogen specific. It is possible that the same mechanisms responsible for the initial recognition of viral infection and induction of IL-10 continue to function throughout persistent infection. On the other hand (but certainly excluding the latter), prolonged/elevated viral levels may trigger subsequent factors that serve to continually stimulate IL-10 production.

As discussed in the introduction, the innate immune system initially senses viral infection via pattern recognition receptors (including multiple TLRs) leading to type-I interferon production and activation of the immune response. However, TLR signaling also induces counter-regulatory molecules, including IL-10. Components of HCV, CMV, EBV and LCMV all bind to TLR2 and TLR2 in turn can induce IL-10 expression via recruitment of the signaling adaptor MyD88 and activation of ERK pathways. In humans, HIV glycoprotein binding to a mannose C-type lectin receptor (likely DC-SIGN) on the surface of monocyte derived-DC led to IL-10 expression. In addition to stimulating IL-10, HIV and LCMV infections also lead to dysregulated type-I interferon production promoting a suppressive environment and further dampening the antiviral response. This is also true for HCV, where interaction of the core protein with TLR2 results not only in up-regulation of IL-10 expression but also decreased expression of type-I interferon by Kupffer cells and pDC. While a second HCV protein, NS3, concurrently up-regulated IL-10 and down-regulated IL-12 expression by macrophages and DC leading to diminished T cell stimulatory capacity in vitro. Therefore increased/prolonged levels of antigen may continue to trigger these same innate receptors throughout infection leading to sustained IL-10 expression while simultaneously down-modulating stimulatory factors and potentiating the immunosuppressive environment.

Continued viral infection also stimulates the de novo expression of factors that potentially modulate IL-10 expression. PD-1/PD-L1 interaction suppresses antiviral T cell activity during persistent virus infection, and has also been shown to increase IL-10 expression. A recent study demonstrated that peripheral blood monocytes from HIV viremic individuals express high levels of PD-1. Stimulation of PD-1 with antibody or PD-L1 transfected cells induced IL-10 expression capable of limiting CD4 T cell proliferation in vitro. These data demonstrate that PD-1 stimulation can activate IL-10 expression and suppression of antiviral immunity. Interestingly, during persistent LCMV infection we observed similar levels of IL-10 RNA expression in wild-type and PD-L1 KO mice indicating that PD-L1 and IL-10 largely interact via different pathways. Functioning through different suppressive pathways was also consistent with the ability of dual IL-10 and PD-L1 blockade to additively increase exhausted T cell function compared to either IL-10R or PD-L1 blockade alone. The difference between these studies may relate to the fact that although most of the monocytes express PD-1, very few produced IL-10 upon PD-1 triggering. As a result, in PD-L1 KO mice the amount of IL-10 triggered by PD-1 on monocytes may not substantially impact the overall level of IL-10 expression. On the other hand, although only a small fraction of monocytes were stimulated to produce IL-10 by PD-1stimulation, these cells may be functionally distinct from other monocyte subsets and therefore, may have an enhanced ability to affect T cell immunity while not contributing significantly to global IL-10 production. Thus, it remains to be determined whether the population of IL-10 producing monocytes in vivo impact CD4 T cell responses similarly to that observed in vitro.

We and others also recently identified the important and progressive role of IL-21 in sustaining CD8 T cell responses during prolonged periods of virus replication. For us, these experiments were initiated in our effort to define the mechanism(s) that induce IL-10 during LCMV persistence and based on the known role of IL-21 in stimulating IL-10 expression. However, no change in IL-10 expression was observed in mice lacking IL-21R expression. Further, we have not observed changes in IL-10 RNA or serum protein expression during persistent LCMV infection in mice deficient in factors that stimulate IL-10 in other models of disease (E. Wilson and D. Brooks, unpublished observations), including IL-27 and Galectin-1 , TLR2 and MyD88. In total the discrepancy between IL-10 inducing factors in other disease models compared to persistent virus infection again indicates that multiple regulatory mechanisms can be instituted to suppress immunity in a pathogen/disease specific manner.

Another mechanism of IL-10 induction may not result specifically from alterations in factors produced, but instead changes in antigen presenting cell subsets. One of the defining characteristics of persistent LCMV variants is their ability to bind with high affinity to their cellular receptor α-dystroglycan enabling efficient infection of dendritic cells . Due to infection, DC become targets for CTL lysis and the loss of DC was associated with the ensuing immunosuppression. In particular, the CD8α+ DC subset is depleted during persistent LCMV infection and the remaining CD8α− DC were shown to increase IL-10 production by virus-specific CD4 T cells, which might in turn suppress antiviral CD8 T cell responses. The preferential killing of mature DC during HIV infection by NK cells in an IL-10 dependent fashion would similarly increase the frequency of immature DC and potentially augment T cell responses. Further, MCMV disruption of APC function leads to insufficient T cell activation, but the decreased levels of MHC may also prevent DC interaction with T cells, thereby effectively shifting the APC subsets that prime/sustain T cells. In reality, it is likely a culmination of all these events (and more yet to be discovered) that account for IL-10 mediated immune suppression to persistent virus infection.


The sum of all the IL-10 induced events (in conjunction with those induced by other suppressive factors) act in concert to orchestrate immune suppression and facilitate viral persistence. Therapeutically, the targeting of multiple cell types by IL-10 means that alleviating IL-10 mediated immunosuppression would enhance several immune parameters compromised by persistent infection.

In HIV infected individuals, IL-10 produced by PBMC inhibits CD4 and CD8 T cell proliferation and cytokine production and blockade of IL-10 efficiently restores these functions in vitro. Interaction of HIV with DC stimulates IL-10 production resulting in multiple functional defects. Interestingly, immature and mature DC respond differently to HIV-induced IL-10 upregulation. Immature DC exhibit an aberrant resistance to NK cell mediated cytolysis, whereas mature DC are targeted and destroyed by DC. This APC switch results in an over represented presence of ‘toleregenic’ DC during HIV infection that may fail to sustain T cell responses and/or ineffectively prime de novo T cell responses against evolving HIV mutants. There is also evidence that IL-10 augments B cell responses during HIV infection, inducing B cell exhaustion that may hinder antibody production. In some circumstances IL-10 is a positive regulator of CD8 T cells. In these situations IL-2 is required for the stimulatory effect of IL-10 on CD8 T cells. However, IL-2 production by CD4 and CD8 T cells is rapidly lost during persistent infections. Thus, the presence of IL-10 without IL-2 may lead to suppressive instead of stimulatory CD8 T cell programming and highlights the important interplay between stimulatory and suppressive factors that fine tune the immune response to affect the outcome of infection.

We and others have clearly established the dominant role of IL-10 in facilitating LCMV persistence and based on its translatability to human persistent viral infections; it is likely that LCMV will be an important system to address IL-10 induced immunosuppression. IL-10 is produced by multiple APC subsets during persistent LCMV infection. Although priming of virus-specific CD4 and CD8 T cells is initially effective during persistent infection, the subsequent interactions with APC (i.e., occurring after the initial priming events) may attenuate ongoing T cell responses. CD4 T cell help is critical during persistent LCMV infection to sustain antiviral immune responses. IL-10 directly targets CD4 T cells during an acute LCMV infection and similar diminution/alteration of the CD4 response by IL-10 during viral persistence may attenuate help, further exasperating the debilitated immune response. IL-10 may also be acting directly on virus-specific CD8 T cells, B cells and/or NK cells to attenuate their function and facilitate viral persistence. Identification of the relevant targets of IL-10 in vivo are currently underway and should yield important insight into the mechanisms that abort immune responses to facilitate viral persistence."[3]


Targeting ImmunoSupression - Blocking IL-10

"The initial finding that IL-10R blockade prevented T cell exhaustion and facilitated immune-mediated eradication of an otherwise persistent LCMV infection was the first example that single factor could alone suppress antiviral immunity to prevent virus clearance. In addition to early blockade of IL-10 to prevent T cell exhaustion and LCMV persistence, late blockade of IL-10 activity also enhanced T cell responses leading to control of an established persistent infection. These findings indicate that IL-10 suppresses and can be targeted to restore antiviral immunity at multiple stages throughout persistent infection. Similarly, IL-10R blockade prevents MCMV persistence, although in the latter case the enhanced antiviral effects were accompanied by increased immunopathology."[3]


"Elevated serum levels of IL-10 are observed during many persistent virus infections in humans and similar to LCMV infection, correlates with diminished T cell activity and increased virus replication. Blockade of IL-10 in vitro restores function to HIV-specific and HCV-specific T cells. Consistent with the correlation between HIV replication and IL-10 expression, antibody blockade of IL-10 only increased T cell function when cells were isolated from productively infected individuals and had minimal impact when cells originating from patients with effectively suppressed levels of HIV replication were analyzed. However, in a separate study IL-10 blockade was shown to efficiently boost T cell responses even during effective anti-HIV therapy (in which HIV replication and IL-10 expression were low). In total, these multiple studies demonstrate that blocking IL-10 during HIV infection can enhance antiviral T cell responses, but importantly, they indicate that IL-10 differentially affects different individuals and does so at distinct phases of infection.

The tight relationship between IL-10 and virus titers suggests that IL-10 may serve as a rheostat to constantly modulate immunity in relation to changing levels of virus replication. In agreement with this function, the experiments in which IL-10 was blocked to enhance HIV and HCV specific responses were performed after cell isolation techniques that eliminated in vivo produced IL-10. These findings indicate that de novo IL-10 production continually suppress T cell responses- T cells that are teetering on a fine line between exhaustion and productive immunity. Once function is diminished, blockade of no single factor restores T cell activity to that observed during an acute infection. However, therapies that alleviate some level of suppression appear to propel T cells across that fine line to better fight infection.

Recent research has clearly established that multiple negative immune-regulatory mechanisms are invoked to suppress T cell responses during viral persistence. We recently demonstrated that at least two of these factors (i.e., IL-10 and PD-L1) operate through distinct pathways to suppress immunity. As a result, dual antibody blockade of IL-10 and PD-L1 during LCMV persistence significantly enhanced antiviral T cell responses compared to blockade of either factor alone and rapidly controlled systemic viral replication. Similarly, simultaneous blockade of PD-L1 and another inhibitory receptor Lag-3, further enhanced T cell responses despite Lag-3 blockade alone having only minimal effect. This is particularly important because it indicates that there are layers of immunosuppression and that some negative regulatory pathways are dominant over others during persistent infection. However, once the dominant suppression is relieved other factors that appear to have limited function may become relevant and serve as targets to additionally enhance antiviral immunity.

Due to the varied lifecycles of persistent viruses, it will ultimately be important to determine how IL-10 blockade (as well as blockade of other negative regulatory factors) impacts control of different viral infections. HIV rapidly establishes a long-lived latent reservoir that is able to rekindle infection after prolonged periods of virus control. During latent infection HIV remains hidden from immune recognition and as a result would not be targeted by therapies that amplify immune fundamentals. However in cases like HCV infection where a latent viral reservoir is not established and therapeutic elimination of infection can be achieved, overcoming exhaustion and boosting immunity by blocking IL-10 activity could further control HCV replication. Thus, blockade of IL-10 (or other regulatory factors) may facilitate control (perhaps even long-term control) over HIV infection, but may not be able to completely eradicate infection because of immune-resistant latent reservoirs. On the other hand, a similar blockade of IL-10 during HCV infection may ultimately facilitate long term clearance of infection due to the absence of a long-lived latent reservoir.

IL-10 also regulates immunity to acute viral infections, limiting the magnitude of the ensuing response, the production of effector cytokines and consequently immunopathology, but generally without substantially impacting viral titers or clearance kinetics. Infection of mice with Influenza often leads to severe immunopathology, morbidity and mortality and IL-10 limits these negative effects. Following influenza infection of mice, a large population of lung infiltrating CD4 and particularly CD8 T cells produce IL-10. Blockade of IL-10 enhanced IFNγ production leading to increased immunopathology and mortality without impacting virus clearance. These data suggest that in response to unknown cues, virus-specific CD8 T cells are capable of producing IL-10 to curb their own responses. IL-10 expression also restricted CD4 T cell responses during influenza resulting in decreased antibody titers; whereas, lack of IL-10 expression led to increased influenza specific antibody production and enhanced survival. Interestingly, IL-10 mediated immunosuppression was linked to heightened susceptibility to secondary bacterial infection following influenza infection although many factors likely contribute. IL-10 is rapidly upregulated following acute LCMV-Armstrong infection and although not to the extent observed following persistent LCMV infection, it negatively regulated what is generally considered the ‘optimal’ antiviral immune response. Interestingly, blockade of IL-10 activity directly enhanced both the quality and the quantity of virus-specific CD4 T cell responses without affecting virus-specific CD8 T cells, illustrating the differential regulation of T cell subsets following infection. Minor decreases in virus titers were observed in acute LCMV infected, IL-10 deficient mice, although both wild-type and IL-10 deficient mice cleared virus with a comparable kinetic. Excitingly, this promiscuity of IL-10 further substantiates the ability to restore many diverse effector mechanisms by blocking a single molecule. The increased expression and inhibitory activity of IL-10 following acute viral infection suggest that IL-10 blockade may be an effective adjuvant to prophylactic (i.e., preventative) vaccines to further enhance immunity. This would be particularly true for vaccines in which heightened CD4 T cell responses would be beneficial, such as HCV wherein the strength of the CD4 T cell response is an important determinant of clearance.

Unlike prophylactic vaccines that aim to engender immune memory de novo, therapeutic vaccines (i.e., vaccines delivered during an established viral infection) must rebuild a debilitated immune response to now overcome the infection that it could not initially control. Along this line, vaccine agents that are immunogenic when administered to antigen naïve individuals often fail to efficiently stimulate immunity when provided prophylactically. Additionally, many prophylactic vaccines rely on stimulating antibody production, whereas therapeutic vaccines will likely have to restore/stimulate antiviral T cell responses as well as other immune parameters that are often refractory to further stimulation. The finding that IL-10 actively inhibited T cell responses during persistent infection led us to hypothesize that one reason therapeutic vaccination strategies have thus far failed to resurrect/sustain T cell responses and control persistent infection is because they do not alleviate the immunosuppressive environment. Consequently, even if T cell responses could be restored they would rapidly again succumb to the same constraints that had previously limited their responsiveness. Consistent with this mechanism we demonstrated that antibody blockade of IL-10 during an established persistent viral infection permitted an otherwise ineffective DNA vaccine now highly efficient at stimulating CD4 and CD8 T cell responses leading to accelerated clearance of the persistent infection. In conjunction, Rafi Ahmed’s group demonstrated that during persistent LCMV infection PD-L1 blockade similarly enhanced therapeutic vaccination with a live-replicating vaccine vector. Together, our findings established the immunosuppressive environment as an important factor inhibiting vaccination attempts to restore antiviral T cell function during persistent viral infection and suggested that blockade of negative immune regulatory molecules may ultimately prove a powerful strategy to aid therapeutic vaccination and purge an established persistent viral infection."[3]


Next....

"The initial discovery of IL-10 as an inhibitor of Th1 differentiation has rapidly diversified such that now IL-10 is widely considered a ‘master-regulator’ of host immunity. The conserved nature of IL-10 mediated suppression among evolutionarily distinct species and the ability to boost immune function by blocking a single factor, despite the presence of other very powerful negative immune regulators, is quite astounding and speaks clearly to the significance of this pathway. However, many important discoveries remain to be made concerning IL-10 mediated suppression and how best to manipulate it for therapeutic benefit. The promiscuity of IL-10 production and function suggests that its blockade could amplify multiple antiviral mechanisms to control persistent virus replication. By understanding how IL-10 regulates distinct components of the immune response it may be possible to block IL-10 production by or function on certain cells and unleash antiviral T cells while maintaining regulation of those cells prevent immunopathology. Ultimately, blockade of immune-regulatory factors holds great promise as an approach to restore immunity and purge established persistent viral infections. The ability of IL-10 and other inhibitory factors to operate at distinct levels of immune function and on different cell subsets indicates the possibility of combinatorial blockade cocktails to specifically enhance desired immune cell subsets and evoke different immune responses; thus, paving the way into an age of rationale vaccine design."[3]


References:

  1. Virus-Encoded Homologs of Cellular Interleukin-10 and Their Control of Host Immune Function. Authors: Barry Slobedman barry_slobedman@wmi.usyd.edu.au, Peter A. Barry, Juliet V. Spencer ASM Journals. Journal of Virology. Vol. 83, No. 19. Minireview

  2. Mini-review The paradoxical role of IL-10 in immunity and cancer Mark H. Mannino a , Ziwen Zhu. Cancer Letters (2015) By Mark H. Mannino, et al., The paradoxical role of IL-10 in immunity and cancer, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.07.009 Contents lists available at ScienceDirect Cancer Letters.

  3. The role of IL-10 in regulating immunity to persistent viral infections. Curr Top Microbiol Immunol. Author manuscript; available in PMC 2012 Nov 7. Curr Top Microbiol Immunol. 2011; 350: 39–65. doi: 10.1007/82_2010_96. PMCID: PMC3492216. NIHMSID: IHMS417038. PMID: 20703965

  4. A Potential Role of Interleukin 10 in COVID-19 Pathogenesis. Ligong Lu,Hui Zhang, et. al. Trends Immunol. 2021 Jan; 42(1): 3–5. Published online 2020 Nov 2. doi: 10.1016/j.it.2020.10.012. PMCID: PMC7605819. PMID: 33214057




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