Granulocyte macrophage colony stimulating factor exerts dominant effects over macrophage colony stimulating factor during macrophage differentiation in vitro to induce an inflammatory phenotype.

BACKGROUND: Macrophages (Mφ) can exist along a spectrum of phenotypes that include pro-inflammatory (M1) or anti-inflammatory (M2) immune cells. Mφ colony stimulating factor (M-CSF) and granulocyte Mφ colony stimulating factor (GM-CSF) are cytokines important in hematopoiesis, polarization and activation of Mφ. METHODS AND RESULTS: To gain a greater understanding of the relationship between GM-CSF and M-CSF, we investigated an in vitro model of differentiation to determine if GM-CSF and M-CSF can antagonize each other, in terms of Mφ phenotype and functions. We determined that Mφ cultured in mixed M-CSF: GM-CSF ratios exhibit M1-like GM-CSF-treated macrophage phenotype when the ratios of the two cytokines are 1:1 in culture. Moreover, GM-CSF is dominant over M-CSF in influencing Mφ production of proinflammatory cytokines such as IL-6, TNFα, and IL-12p40, and the anti-inflammatory cytokine IL-10. CONCLUSIONS: Our data established that GM-CSF is more dominant over M-CSF, triggering the Mφ to become pro-inflammatory cells. These findings provide insight into how GM-CSF can influence Mφ activation with implications in inflammatory diseases where the Mφ status can play a significant role in supporting the inflammatory conditions.

IFN-ß activates cytotoxic function of human natural killer cells toward IL-27 and poly(I:C) stimulated PC3 and DU145 cells.

Natural killer (NK) cell phenotype and function are altered in patients with prostate cancer, and increased NK cell activity is associated with a better prognosis in patients with disease. For patients with advanced stage prostate cancer, immunotherapies are a promising approach when standard treatment options have been exhausted. With the rapid emergence of NK cell-based therapies, it is important to understand the mechanisms by which NK cells can be triggered to kill cancer cells that have developed immune-evasive strategies. Altering the cytokine profiles of advanced prostate cancer cells may be an area to explore when considering ways in which NK cell activation can be modulated. We have previously demonstrated that combining the cytokine, IL-27, with TLR3 agonist, poly(I:C), changes cytokine secretion in the advanced prostate cancer models, PC3 and DU145 cells. Herein, we extend our previous work to study the effect of primary human NK cells on prostate cancer cell death in an in vitro co-culture model. Stimulating PC3 and DU145 cells with IL-27 and poly(I:C) induced IFN-ß secretion, which was required for activation of primary human NK cells to kill these stimulated prostate cancer cells. PC3 cells were more sensitized to NK cell-mediated killing when compared to DU145 cells, which was attributed to differential levels of IFN-ß produced in response to stimulation with IL-27 and poly(I:C). IFN-ß increased granzyme B secretion and membrane-bound TRAIL expression by co-cultured NK cells. We further demonstrated that these NK cells killed PC3 cells in a partially TRAIL-dependent manner. This work provides mechanistic insight into how the cytotoxic function of NK cells can be improved to target cancer cells.

Medical Devices, Invisible Women, Harmful Consequences.

In this commentary, we explore the disproportionate risk women experience with the insertion of various medical devices. Although pre-market device testing and complication tracking could be improved for all, a failure to consider sex differences in hormones, anatomy, inflammatory responses, and physical function puts women at particular risk. This invisibility of women is an example of gender bias in medical science and practice, a bias that could be corrected in the ways we suggest.

Activation of TLR4 by viral glycoproteins: A double-edged sword?

Recognition of viral infection by pattern recognition receptors is paramount for a successful immune response to viral infection. However, an unbalanced proinflammatory response can be detrimental to the host. Recently, multiple studies have identified that the SARS-CoV-2 spike protein activates Toll-like receptor 4 (TLR4), resulting in the induction of proinflammatory cytokine expression. Activation of TLR4 by viral glycoproteins has also been observed in the context of other viral infection models, including respiratory syncytial virus (RSV), dengue virus (DENV) and Ebola virus (EBOV). However, the mechanisms involved in virus-TLR4 interactions have remained unclear. Here, we review viral glycoproteins that act as pathogen-associated molecular patterns to induce an immune response via TLR4. We explore the current understanding of the mechanisms underlying how viral glycoproteins are recognized by TLR4 and discuss the contribution of TLR4 activation to viral pathogenesis. We identify contentious findings and research gaps that highlight the importance of understanding viral glycoprotein-mediated TLR4 activation for potential therapeutic approaches.

IL-27 Improves Prophylactic Protection Provided by a Dead Tumor Cell Vaccine in a Mouse Melanoma Model.

The protocol used to induce cell death for generating vaccines from whole tumor cells is a critical consideration that impacts vaccine efficacy. Here we compared how different protocols used to induce cell death impacted protection provided by a prophylactic whole tumor cell vaccine in a mouse melanoma model. We found that melanoma cells exposed to γ-irradiation or lysis combined with UV-irradiation (LyUV) provided better protection against tumor challenge than lysis only or cells exposed to UV-irradiation. Furthermore, we found that the immunoregulatory cytokine, IL-27 enhanced protection against tumor growth in a dose-dependent manner when combined with either LyUV or γ-irradiated whole tumor cell vaccine preparations. Taken together, this data supports the use of LyUV as a potential protocol for developing whole tumor cell prophylactic cancer vaccines. We also showed that IL-27 can be used at low doses as a potent adjuvant in combination with LyUV or γ-irradiation treated cancer cells to improve the protection provided by a prophylactic cancer vaccine in a mouse melanoma model.

Antiviral Activities of Interleukin-27: A Partner for Interferons?

Emergence of new, pandemic-level viral threats has brought to the forefront the importance of viral immunology and continued improvement of antiviral therapies. Interleukin-27 (IL-27) is a pleiotropic cytokine that regulates both innate and adaptive immune responses. Accumulating evidence has revealed potent antiviral activities of IL-27 against numerous viruses, including HIV, influenza, HBV and more. IL-27 contributes to the immune response against viruses indirectly by increasing production of interferons (IFNs) which have various antiviral effects. Additionally, IL-27 can directly interfere with viral infection both by acting similarly to an IFN itself and by modulating the differentiation and function of various immune cells. This review discusses the IFN-dependent and IFN-independent antiviral mechanisms of IL-27 and highlights the potential of IL-27 as a therapeutic cytokine for viral infection.

Sustained IL-4 priming of macrophages enhances the inflammatory response to TLR7/8 ligand R848.

Macrophages (Mϕ) are highly plastic, and can acquire a variety of functional phenotypes depending on the presence of different stimuli in their local environment. Mφ stimulated by interleukin (IL)-4 induce an alternative activation state and function as anti-inflammatory cells and promote tissue repair. However, there is overwhelming evidence that IL-4 can play a role in promoting inflammation. In asthma and allergic inflammation, IL-4 mediates proinflammatory responses that lead to tissue damage. Thus the effect of IL-4 on the outcome of the immune responses is greatly influenced by other cofactors and cytokines present in the microenvironment. R848 (resiquimod), a TLR7/8 agonist is a novel vaccine adjuvant, triggering a strong Th1-skewed response but its efficacy as a vaccine adjuvant shows variable results. It is not currently known whether the presence of IL-4 can dampen or enhance immunity in response to TLR7 agonists. In the present study, we sought to investigate the impact of IL-4-induced Mφ polarization on the outcome of R848 stimulation. The activation marker expression and production of cytokines were measured in murine spleen-derived Mφ. Protein expression levels of innate recognition molecules and transcription factors involved, including retinoic-acid inducible gene I, mitochondrial antiviral signaling protein, stimulator of interferon genes (STING), and IFN regulatory factors were evaluated in activated Mφ. These play a crucial role in the control of viral replication and optimal CD8+ T cell priming. We report that sustained priming with IL-4 alone promotes an antiviral response in Mφ, and enhances proinflammatory responses to R848 treatment. This highlights the need for better understanding of IL-4 proinflammatory functions and its potential use as a broad-acting antiviral in combination with R848 may be used in combination with other therapies to target the innate arm of immunity against emerging infections.

TLR7 Ligation Inhibits TLR8 Responsiveness in IL-27-Primed Human THP-1 Monocytes and Macrophages.

Regulation of proinflammatory cytokine expression is critical in the face of single-stranded RNA (ssRNA) virus infections. Many viruses, including coronavirus and influenza virus, wreak havoc on the control of cytokine expression, leading to the formation of detrimental cytokine storms. Understanding the regulation and interplay between inflammatory cytokines is critical to the identification of targets involved in controlling the induction of cytokine expression. In this study, we focused on how the antiviral cytokine interleukin-27 (IL-27) regulates signal transduction downstream of Toll-like receptor 7 (TLR7) and TLR8 ligation, which recognize endosomal single-stranded RNA. Given that IL-27 alters bacterial-sensing TLR expression on myeloid cells and can inhibit replication of single-stranded RNA viruses, we investigated whether IL-27 affects expression and function of TLR7 and TLR8. Analysis of IL-27-treated THP-1 monocytic cells and THP-1-derived macrophages revealed changes in mRNA and protein expression of TLR7 and TLR8. Although treatment with IL-27 enhanced TLR7 expression, only TLR8-mediated cytokine secretion was amplified. Furthermore, we demonstrated that imiquimod, a TLR7 agonist, inhibited cytokine and chemokine production induced by a TLR8 agonist, TL8-506. Delineating the immunomodulatory role of IL-27 on TLR7 and TLR8 responses provides insight into how myeloid cell TLR-mediated responses are regulated during virus infection.

Differential TLR7-mediated cytokine expression by R848 in M-CSF- versus GM-CSF-derived macrophages after LCMV infection.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) play an important role in macrophage (MФ) development by influencing their differentiation and polarization. Our goal was to explore the difference between M-CSF- and GM-CSF-derived bone marrow MФ responsiveness to TLR7-mediated signalling pathways that influence cytokine production early after infection in a model of acute virus infection. To do so, we examined cytokine production and TLR7-mediated signalling at 1 h post-lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) infection. We found that R848-induced cytokine expression was enhanced in these cells, with GM-CSF cells exhibiting higher proinflammatory cytokine expression and M-CSF cells exhibiting higher anti-inflammatory cytokine expression. However, R848-mediated signalling molecule activation was diminished in LCMV-infected M-CSF and GM-CSF macrophages. Interestingly, we observed that TLR7 expression was maintained during LCMV infection of M-CSF and GM-CSF cells. Moreover, TLR7 expression was significantly higher in M-CSF cells compared to GM-CSF cells. Taken together, our data demonstrate that although LCMV restrains early TLR7-mediated signalling, it primes differentiated MФ to enhance expression of their respective cytokine profiles and maintains levels of TLR7 expression early after infection.

Granulocyte/Macrophage Colony-Stimulating Factor-Derived Macrophages Exhibit Distinctive Early Immune Response to Lymphocytic Choriomeningitis Virus Infection.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF) modulate differentiation and immune functions of macrophages (MΦ). Our aim was to evaluate how different MΦ differentiation conditions influence the MΦ response to virus infection. To address this, we differentiated bone marrow-derived MΦ in either GM-CSF or M-CSF and measured the cytokine responses to two different strains of lymphocytic choriomeningitis virus (LCMV) (clone 13; Cl13 or Armstrong; ARM). GM-CSF MΦ infected with either LCMV-ARM or -Cl13 produced more IL-6 than M-CSF MΦ, whereas M-CSF MΦ generated more IL-10 than GM-CSF MΦ. Interestingly, in M-CSF MΦ, LCMV-ARM induced more IL-10 production than Cl13. However, we could not detect any IL-12p70 or IL-23 after infection from either cell types. We also observed that GM-CSF MΦ was more efficient than M-CSF MΦ in supporting antigen-specific CD8+ T cell proliferation. Taken together, our data demonstrate that GM-CSF and M-CSF MΦ differ in how they respond to viral infection by their production of different cytokines, and their support for CD8+ T cell proliferation.

Who's in charge here? Macrophage colony stimulating factor and granulocyte macrophage colony stimulating factor: Competing factors in macrophage polarization.

Macrophages make up a crucial aspect of the immune system, carrying out a variety of functions ranging from clearing cellular debris to their well-recognized roles as innate immune cells. These cells exist along a spectrum of phenotypes but can be generally divided into proinflammatory (M1) and anti-inflammatory (M2) groups, representing different states of polarization. Due to their diverse functions, macrophages are implicated in a variety of diseases such as atherosclerosis, lupus nephritis, or infection with HIV. Throughout their lifetime, macrophages can be influenced by a wide variety of signals that influence their polarization states, which can affect their function and influence their effects on disease progression. This review seeks to provide a summary of how GM-CSF and M-CSF influence macrophage activity during disease, and provide examples of in vitro research that indicate competition between the two cytokines in governing macrophage polarization. Gaining a greater understanding of the relationship between GM-CSF and M-CSF, along with how these cytokines fit into the larger context of diseases, will inform their use as treatments or targets for treatment in various diseases.

IL-27, IL-30, and IL-35: A Cytokine Triumvirate in Cancer.

The role of the immune system in anti-tumor immunity cannot be overstated, as it holds the potential to promote tumor eradication or prevent tumor cell escape. Cytokines are critical to influencing the immune responses and interactions with non-immune cells. Recently, the IL-12 and IL-6 family of cytokines have accumulated newly defined members each with specific immune functions related to various cancers and tumorigenesis. There is a need to better understand how cytokines like IL-27, IL-30, and IL-35 interact with one another, and how a developing tumor can exploit these interactions to enhance immune suppression. Current cytokine-based immunotherapies are associated with cytotoxic side effects which limits the success of treatment. In addition to this toxicity, understanding the complex interactions between immune and cancer cells may be one of the greatest challenges to developing a successful immunotherapy. In this review, we bring forth IL-27, IL-30, and IL-35, "sister cytokines," along with more recent additions to the IL-12 family, which serve distinct purposes despite sharing structural similarities. We highlight how these cytokines function in the tumor microenvironment by examining their direct effects on cancer cells as well their indirect actions via regulatory functions of immune cells that act to either instigate or inhibit tumor progression. Understanding the context dependent immunomodulatory outcomes of these sister cytokines, as well as their regulation within the tumor microenvironment, may shed light onto novel cancer therapeutic treatments or targets.

Poly(I:C)-Mediated Death of Human Prostate Cancer Cell Lines Is Induced by Interleukin-27 Treatment.

Interleukin (IL)-27 is a promising anti-cancer cytokine with therapeutic potential. Exhibiting overlapping properties with type I and II interferons (IFNs), IL-27 impacts cancer cell viability and immune cell activity. Known to modulate toll-like receptor (TLR) expression, we investigated whether IL-27 affected TLR-mediated death in cancer cells. Using DU145 and PC3 cell lines as models of prostate cancer, we investigated whether IL-27 and IFN-γ affect TLR3-mediated cell death. Our results demonstrate that when IL-27 or IFN-γ is added with polyinosinic-polycytidylic acid [poly(I:C)], type I IFN (IFN-I) expression increases concurrently with cell death. IL-27 and IFN-γ enhanced TLR3 expression, suggesting a mechanism for sensitization to cell death. Further, PC3 cells were more sensitive to IL-27/poly(I:C)-induced cell death compared with DU145 cells. This correlated with higher production of IFN-ß and inducible protein-10 versus IL-6 in response to treatment of PC3 cells compared with DU145. Taken together, this study demonstrates a potential role for IL-27 in the treatment of prostate cancer.

Activation of Peripheral Blood CD4+ T-Cells in IBS is not Associated with Gastrointestinal or Psychological Symptoms.

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS), but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight, we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin ß7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore, phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFα secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFα production, while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms, but not markers of immune activation, decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation, but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms.

The effects of CD14 and IL-27 on induction of endotoxin tolerance in human monocytes and macrophages.

Upon repeated exposure to endotoxin or lipopolysaccharide (LPS), myeloid cells enter a refractory state called endotoxin tolerance as a homeostatic mechanism. In innate immune cells, LPS is recognized by co-receptors Toll-like receptor 4 (TLR4) and CD-14 to initiate an inflammatory response for subsequent cytokine production. One such cytokine, interleukin (IL)-27, is produced by myeloid cells in response to bacterial infection. In monocytes, IL-27 has proinflammatory functions such as up-regulating TLR4 expression for enhanced LPS-mediated cytokine production; alternatively, IL-27 induces inhibitory functions in activated macrophages. This study investigated the effects of IL-27 on the induction of endotoxin tolerance in models of human monocytes compared with macrophages. Our data demonstrate that IL-27 inhibits endotoxin tolerance by up-regulating cell surface TLR4 expression and soluble CD14 production to mediate stability of the surface LPS-TLR4-CD14 complex in THP-1 cells. In contrast, elevated basal expression of membrane-bound CD14 in phorbol 12-myristate 13-acetate (PMA)-THP-1 cells, primary monocytes, and primary macrophages may promote CD14-mediated endocytosis and be responsible for the preservation of an endotoxin-tolerized state in the presence of IL-27. Overall, the efficacy of IL-27 in inhibiting endotoxin tolerance in human THP-1 monocytes and PMA-THP-1 macrophages is affected by membrane-bound and soluble CD14 expression.

Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes.

Interleukin (IL)-30, the IL-27p28 subunit of the heterodimeric cytokine IL-27, acts as an antagonist of IL-27 and IL-6 signaling in murine cells via glycoprotein 130 (gp130) receptor and additional binding partners. Thus far, functions of IL-30 have not been fully elucidated in human cells. We demonstrate that like IL-27, IL-30 upregulated TLR4 expression to enhance lipopolysaccharide-induced TNF-α production in human monocytes; however, these IL-30-mediated activities did not reach the same levels of cytokine induction compared to IL-27. Interestingly, IL-30- and IL-27-mediated interferon-γ-induced protein 10 (IP-10) production required WSX-1 engagement and signal transducer and activator of transcription (STAT) 3 phosphorylation; furthermore, IL-30 induced STAT phosphorylation after 16 h, whereas IL-27 induced STAT phosphorylation within 30 min. This prompted us to examine if a secondary mediator was required for IL-30-induced pro-inflammatory functions, and hence we examined IL-6-related molecules. Combined with inhibition of soluble IL-6 receptor α (sIL-6Rα) and data showing that IL-6 inhibited IL-30/IL-27-induced IP-10 expression, we demonstrate a role for sIL-6Rα and gp130 in IL-30-mediated activity in human cells.

Lymphocytic choriomeningitis virus infection of dendritic cells interferes with TLR-induced IL-12/IL-23 cytokine production in an IL-10 independent manner.

Dendritic cells produce IL-12 and IL-23 in response to viral and bacterial infection and these cytokines are responsible for successful pathogen clearance. How sequential viral and bacterial infections affect the production of IL-12 and IL-23 is currently not known. Our study demonstrates that in dendritic cells infected with Lymphocytic choriomeningitis virus (LCMV), TLR activation with bacterial PAMPs resulted in reduced IL-12 and IL-23 expression compared to non-infected cells. Furthermore, expression of other proinflammatory cytokines, TNF-α and IL-6, were not inhibited under these conditions. We discovered that TLR-induced phosphorylation of p38 was significantly inhibited in LCMV-infected cells. We detected enhanced expression of suppressor of cytokine signalling (SOCS)-3 and IL-10. Yet, neutralizing IL-10 did not restore IL-12/IL-23 expression. Taken together, these results show that virus infection interferes with the magnitude of TLR-mediated inflammatory responses by repressing specific cytokine expression.

On taking the STING out of immune activation.

Nearly a decade ago, an endoplasmic reticulum (ER) adaptor protein called stimulator of interferon genes (STING) was found to be critical in the induction of type I IFN production in response to DNA virus infection. STING functions by sensing cytoplasmic DNA and activates key transcription factors, including IFN regulatory factor (IRF)-3 and IRF7, to initiate type I IFN expression. Type I IFNs are vital in immunity against viral infections and can influence cancer cell proliferation, migration, and apoptosis. Several studies have shown that STING activation results in potent antitumor activity by generating strong tumor-specific cytotoxic T-cell responses. Moreover, compared with wild-type, STING-knockout mice show greater susceptibility to viral infections. In this review, we discuss the importance of STING signaling during the induction of immune responses, especially those associated with type I IFN in viral infections and tumor immunity. Furthermore, we highlight recent data that unravel how the STING signaling pathway can be negatively regulated.

The Toll for Trafficking: Toll-Like Receptor 7 Delivery to the Endosome.

Toll-like receptor (TLR)-7 is an endosomal innate immune sensor capable of detecting single-stranded ribonucleic acid. TLR7-mediated induction of type I interferon and other inflammatory cytokine production is important in antiviral immune responses. Furthermore, altered TLR7 expression levels are implicated in various autoimmune disorders, indicating a key role for this receptor in modulating inflammation. This review is focused on the regulation of TLR7 expression and localization compared to that of the other endosomal TLRs: TLR3, 8, and 9. Endosomal TLR localization is a tightly controlled and intricate process with some shared components among various TLRs. However, TLR-specific mechanisms must also be in place in order to regulate the induction of pathogen- and cell-specific responses. It is known that TLR7 is shuttled from the endoplasmic reticulum to the endosome via vesicles from the Golgi. Several chaperone proteins are required for this process, most notably uncoordinated 93 homolog B1 (Caenorhabditis elegans), recently identified to also be involved in the localization of the other endosomal TLRs. Acidification of the endosome and proteolytic cleavage of TLR7 are essential for TLR7 signaling in response to ligand binding. Cleavage of TLR7 has been demonstrated to be accomplished by furin peptidases in addition to cathepsins and asparagine endopeptidases. Moreover, triggering receptor expressed on myeloid cells like 4, a protein associated with antigen presentation and apoptosis in immune cells, has been implicated in the amplification of TLR7 signaling. Understanding these and other molecular mechanisms controlling TLR7 expression and trafficking will give insight into the specific control of TLR7 activity compared to the other endosomal TLRs.