GM-CSF Treatment Research Articles

Abstract 5622: A novel immunocytokine fusion protein combining tumor-targeting anti-CD47 antibody with GM-CSF cytokine for enhanced antitumor efficacy

Abstract Introduction CD47 blockade has emerged as a promising cancer immunotherapy by promoting phagocytosis of tumor cells. However, this treatment strategy may be limited by the scarcity of macrophages with phagocytic potential at the tumor site. The key myeloid cytokine GM-CSF has been approved as an adjunctive agent in the treatment of cancer by virtue of its ability to stimulate hematopoiesis and particularly induce the differentiation of pro-inflammatory M1 macrophages. Here we report the development of a novel immunocytokine composed of a newly discovered CD47 IgG1 fused with GM-CSF at the C-terminus that exhibited enhanced phagocytosis and M1 macrophage activation, leading to superior antitumor efficacy. Methods A lead anti-CD47 mAb was identified from panning of a naïve human library and a series of functional screening assays. The human GM-CSF sequence was then fused via various flexible linkers to the C-terminus of 1F8's heavy chain to generate different formats of anti-CD47-GMCSF fusions. The resultant fusion proteins were tested for retention of anti-CD47- and GM-CSF-mediated biological activities. A lead molecule (1F8-GMCSF) was then tested for its ability to reduce tumor growth in immunodeficient NSG mice. In addition, phenotype of tumor associated macrophages (TAM) was investigated in tumor sections by immunohistochemistry (IHC). Results In macrophage-tumor cell co-culture, treatment with 1F8-GMCSF fusion molecule led to significantly higher levels of tumor cell phagocytosis than that with anti-CD47 alone. Moreover, higher levels of pro-inflammatory cytokines were detected in co-culture supernatant after treatment with 1F8-GMCSF than that with GM-CSF alone. These data suggest functional cooperation between phagocytosis pathways and GM-CSF-mediated growth and differentiation pathways, leading to synergism for enhanced phagocytosis and increased M1 polarization. Consequently, treatment of 1F8-GMCSF in a tumor xenograft model demonstrated superior efficacy in suppression of tumor growth compared to 1F8 or GM-CSF treatment alone or in combination. IHC analysis of tumor sections confirmed a shift from M2 to M1 phenotype in infiltrating macrophages after 1F8-GMCSF treatment. Conclusion We have successfully generated a novel anti-CD47-GM-CSF immunocytokine fusion protein that not only retains key properties of its components but also demonstrates enhanced anti-tumor efficacy and M1 macrophage polarization. This fusion protein represents a second generation anti-CD47 molecule that may be used for treatment of solid tumors and is undergoing preclinical development with an aim to enter clinical studies in 2018. Citation Format: Zhengyi Wang, Wei Cao, Taylor Guo, Jingwu Zang. A novel immunocytokine fusion protein combining tumor-targeting anti-CD47 antibody with GM-CSF cytokine for enhanced antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5622.

Inflammation-Induced Epithelial-to-Mesenchymal Transition and GM-CSF Treatment Stimulate Mesenteric Mesothelial Cells to Transdifferentiate into Macrophages.

In our previous work, we showed that during inflammation-induced epithelial-to-mesenchymal transition (EMT), mesenteric mesothelial cells express ED1 (pan-macrophage marker), indicating that they are transformed into macrophage-like cells. In this paper, we provide additional evidences about this transition by following the phagocytic activity and the TNFα production of mesenteric mesothelial cells during inflammation. Upon injection of India ink particles or fluorescent-labeled bioparticles (pHrodo) into the peritoneal cavity of rats pretreated with Freund's adjuvant, we found that mesothelial cells efficiently engulfed these particles. A similar increase of internalization could be observed by mesothelial cells in GM-CSF pretreated primary mesenteric culture. Since macrophages are the major producers of tumor necrosis factor, TNFα, we investigated expression level of TNFα during inflammation-induced EMT and found that TNFα was indeed expressed in these cells, reaching the highest level at the 5th day of inflammation. Since TNFα is one of the target genes of early growth response (EGR1) transcription factor, playing important role in monocyte-macrophage differentiation, expression of EGR1 in mesothelial cells was also investigated by Western blot and immunocytochemistry. While mesothelial cells did not express EGR1, a marked increase was observed in mesothelial cells by the time of inflammation. Parallel to this, nuclear translocation of EGR1 was shown by immunocytochemistry at the day 5 of inflammation. Caveolin-1 level was high and ERK1/2 became phosphorylated as the inflammation proceeded showing a slight decrease when the regeneration started. Our present data support the idea that under special stimuli, mesenteric mesothelial cells are able to transdifferentiate into macrophages, and this transition is regulated by the caveolin-1/ERK1/2/EGR1 signaling pathway.

Skin-Specific CD301b+ Dermal Dendritic Cells Drive IL-17−Mediated Psoriasis-Like Immune Response in Mice

Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)-committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b+ DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b+ dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent CD11b+ cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b+ subset. Similar to CD301b- population, CD301b+ dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b+ cDC2. Invivo development of CD301b+ cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b+ dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b+ cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b+ cDC2 effectively prevented IL-17-mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b+ dermal cDC2 subset in the skin and its involvement in psoriatic inflammation.

A comparative analysis of longitudinal computed tomography and histopathology for evaluating the potential of mesenchymal stem cells in mitigating radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a debilitating side effect that occurs in up to 30% of thoracic irradiations in breast and lung cancer patients. RIPF remains a major limiting factor to dose escalation and an obstacle to applying more promising new treatments for cancer cure. Limited treatment options are available to mitigate RIPF once it occurs, but recently, mesenchymal stem cells (MSCs) and a drug treatment stimulating endogenous stem cells (GM-CSF) have been investigated for their potential in preventing this disease onset. In a pre-clinical rat model, we contrasted the application of longitudinal computed tomography (CT) imaging and classical histopathology to quantify RIPF and to evaluate the potential of MSCs in mitigating RIPF. Our results on histology demonstrate promises when MSCs are injected endotracheally (but not intravenously). While our CT analysis highlights the potential of GM-CSF treatment. Advantages and limitations of both analytical methods are contrasted in the context of RIPF.

Platelet-Activation Factor Receptor Induces Interleukin 10 Production through STAT3 Activation in Dendritic Cells

The activation of the platelet-activating factor receptor (PAFR) is associated to a suppressor phenotype in macrophages and dendritic cells (DCs). In the present study, we investigated mechanisms underlying the production of the interleukin 10 (IL-10) through PAFR activation in murine DCs. For this purpose, BALB/c mice bone marrow-derived DCs were differentiated by GM-CSF treatment and stimulated with LPS in the presence of the PAFR antagonist WEB2086. Signalling pathways downstream to TLR4 activation were investigated. We found that LPS stimulus induced PAFR ligands generation by DCs, but it did not affect the PAFR expression. The LPS-induced IL-10 production was found to be partially dependent of PAFR, since it was reduced in the presence of WEB2086. The IL-10 production through PAFR activation was independent on CREB and PPARγ, as the treatment with selective inhibitors of these pathways did not affect the IL-10 production. TLR4 adaptor molecules (MyD88 and TRIF) expression, MAPK, or NF-κB (p105/50 and p65 subunits) activation pathways were also excluded, since they were not affected by the treatment with WEB2086. The blockage of PAFR by WEB2086 downregulated the STAT3 (Tyr705) phosphorylation induced by LPS. Additionally, DCs treated with STAT3 inhibitor (S3I-201) showed reduced IL-10 production to the same levels observed in DCs treated with WEB2086. The requirement of STAT3 was confirmed in PAFR-KO DCs, since the STAT3 inhibition did not affect IL-10 production by these cells. Our data show an additional molecular mechanism whereby PAFR contributes to IL-10 production in DCs and support the importance of the PAFR activation in DCs phenotype and function.

Inhibiting tumor growth by GM-CSF treatment, sufficient ϵ-optimality in one-dimensional space

Inhibiting tumor growth by GM-CSF treatment, sufficient ϵ-optimality in one-dimensional space

IL-3 and GM-CSF modulate functions of splenic macrophages in ENU induced leukemia

Leukemia is often accompanied by enhanced susceptibility to infection due to compromised B cell and T cell functions. The alterations in macrophage functions in leukemia remain less investigated. Herein, we examined macrophages for their functions and responsiveness to IL-3 and GM-CSF in ENU-induced leukemia in BALB/c mice. We observed that the macrophages from the leukemic mice had less phagocytic activity, reduced chemotactic activity in response to monocyte chemoattractant protein-1(MCP-1), and decreased expression of iNOS, GM-CSF, TLR2, TLR4, IFN-γ and TNF-α. These impaired macrophage functions in leukemic mice were significantly corrected by IL-3 and GM-CSF treatment indicating the therapeutic benefit of these two cytokines in leukemia.

Upregulation of T-cell Immunoglobulin and Mucin-Domain Containing-3 (Tim-3) in Monocytes/Macrophages Associates with Gastric Cancer Progression

ABSTRACTT-cell immunoglobulin and mucin-domain containing-3 (Tim-3) is an important immune regulatory molecule in cancer immune system. However, expression and function of Tim-3 in monocytes/macrophages in cancer progression mainly remain unclear. In this study, we analyzed Tim-3 levels in peripheral blood mononuclear cells (PBMCs) from 62 gastric cancer patients and 45 healthy controls using flow cytometry and then associated Tim-3 levels with clinical pathological data from patients. We found Tim-3 level was significantly upregulated in monocytes from gastric cancer patients compared with those from healthy controls, and that upregulated Tim-3 levels associated with depth of tumor invasion and tumor lymph node metastasis and advanced clinical stages of gastric cancer patients. Furthermore, tumor-bearing mouse experiments revealed that Tim-3 level on monocytes/macrophages associated with xenograft formation and growth. In addition, culture of monocytes from healthy controls with gastric cancer cell-conditioned medium upregulated Tim-3 expression, but IL-10, TNF-α, IFN-γ, or GM-CSF treatment or T-bet, Eomes, and T-bet/Eomes double gene knockout did not affect Tim-3 levels in blood monocytes/macrophages from human or mouse, respectively. Gal-9/Tim-3 signal was able to significantly stimulate monocyte to secrete IL-6, IL-8, and IL-10, but not IL-1β, IL-12p70, or TNF-α in presence of LPS. In conclusion, our study demonstrated that Tim-3 expressed by monocyte/macrophages might be an important mechanism in gastric cancer progression.

Systemic GM-CSF Recruits Effector T Cells into the Tumor Microenvironment in Localized Prostate Cancer.

Granulocytic-macrophage colony-stimulating factor (GM-CSF) is used as an adjuvant in cancer vaccine trials and has the potential to enhance antitumor efficacy with immunotherapy; however, its immunologic effects are not fully understood. Here, we report results from a phase I study of neoadjuvant GM-CSF in patients with localized prostate cancer undergoing radical prostatectomy. Patients received subcutaneous injections of GM-CSF (250 μg/m2/day) daily for 2 weeks (cohort 1; n = 6), 3 weeks (cohort 2; n = 6), or 4 weeks (cohort 3; n = 6). Treatment was well tolerated with all grade 1 or 2 adverse events. Two patients had a decline in prostate-specific antigen (PSA) of more than 50%. GM-CSF treatment increased the numbers of circulating mature myeloid dendritic cells, proliferating conventional CD4 T cells, proliferating CD8 T cells, and to a lesser magnitude FoxP3+ regulatory CD4 T cells. Although GM-CSF treatment did not augment antigen-presenting cell localization to the prostate, treatment was associated with recruitment of CD8+ T cells to the tumor. These results suggest that systemic GM-CSF can modulate T-cell infiltration in the tumor microenvironment. Cancer Immunol Res; 4(11); 948-58. ©2016 AACR.

Mice Deficient in Angiopoietin-like Protein 2 (Angptl2) Gene Show Increased Susceptibility to Bacterial Infection Due to Attenuated Macrophage Activity

Macrophages play crucial roles in combatting infectious disease by promoting inflammation and phagocytosis. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor that induces tissue inflammation by attracting and activating macrophages to produce inflammatory cytokines in chronic inflammation-associated diseases such as obesity-associated metabolic syndrome, atherosclerosis, and rheumatoid arthritis. Here, we asked whether and how ANGPTL2 activates macrophages in the innate immune response. ANGPTL2 was predominantly expressed in proinflammatory mouse bone marrow-derived differentiated macrophages (GM-BMMs) following GM-CSF treatment relative to anti-inflammatory cells (M-BMMs) established by M-CSF treatment. Expression of the proinflammatory markers IL-1β, IL-12p35, and IL-12p40 significantly decreased in GM-BMMs from Angptl2-deficient compared with wild-type (WT) mice, suggestive of attenuated proinflammatory activity. We also report that ANGPTL2 inflammatory signaling is transduced through integrin α5β1 rather than through paired immunoglobulin-like receptor B. Interestingly, Angptl2-deficient mice were more susceptible to infection with Salmonella enterica serovar Typhimurium than were WT mice. Moreover, nitric oxide (NO) production by Angptl2-deficient GM-BMMs was significantly lower than in WT GM-BMMs. Collectively, our findings suggest that macrophage-derived ANGPTL2 promotes an innate immune response in those cells by enhancing proinflammatory activity and NO production required to fight infection.

GM-CSF treatment prevents respiratory syncytial virus–induced pulmonary exacerbation responses in postallergic mice by stimulating alveolar macrophage maturation

Human respiratory syncytial virus (RSV) is a frequent cause of asthma exacerbations, yet the susceptibility of asthmatic patients to RSV is poorly understood. We sought to address the contribution of resident alveolar macrophages (rAMs) to susceptibility to RSV infection in mice that recovered from allergic airway eosinophilia. Mice were infected with RSV virus after clearance of allergic airway inflammation (AAI). The contribution of post-AAI rAMs was studied invivo by means of clodronate liposome-mediated depletion, adoptive transfer, and treatment with recombinant cytokines before RSV infection. After clearing the allergic bronchial inflammation, post-AAI mice had bronchial hyperreactivity and increased inflammatory cell influx when infected with RSV compared with nonallergic mice, whereas viral clearance was comparable in both mouse groups. Post-AAI rAMs were necessary and sufficient for mediating these proinflammatory effects. In post-AAI mice the residing CD11c(hi) autofluorescent rAM population did not upregulate the terminal differentiation marker sialic acid-binding immunoglobulin-like lectin F and overproduced TNF and IL-6 through increased nuclear factor κB nuclear translocation. In line with these results, post-AAI lungs had reduced levels of the rAM maturation cytokine GM-CSF. Intratracheal administration of GM-CSF induced final rAM maturation in post-AAI mice and prevented the increased susceptibility to RSV-induced hyperreactivity and inflammation. Defective production of GM-CSF leads to insufficient post-AAI rAM maturation in mice that recovered from an AAI, causing increased susceptibility to RSV-induced immunopathology. Promoting the differentiation of post-AAI rAMs might be a therapeutic option for preventing RSV-induced exacerbations in human asthmatic patients.

Whose Gene Is It Anyway? The Effect of Preparation Purity on Neutrophil Transcriptome Studies.

Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of “highly pure” neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.

ID: 25: Substance P as a novel cytokine for induction of alternatively activated M2 polarization and skewing M2 from M1 type of macrophages

We recently showed that intravenous treatment of Substance P (SP) after spinal cord injury increased the number of alternatively activated M2 macrophages at injured lesion of spinal cord and eventually promoted tissue repair. However, it was unclear enhanced M2 polarization of differentiated macrophages is resulted from SP-mediated signaling pathway or accompanied by immune modulatory actions of SP. In the present study, we aim to elucidate the direct effect of SP in M2 polarization in tissue macrophages. Rat macrophages (MO), differentiated with GM-CSF treatment for 5 days on bone marrow-derived monocytes (MO) were prepared. Immunofluorescence staining with specific marker for monocytes and macrophages, MO was characterized as ED-1 (CD68, a marker for macrophages) and CCR7-positive M1-like precursor differentiated from CD14 and CD11b-positive bone marrow monocytes. Administration of SP in MO enhanced the expression of M2 marker, CD206 and CD163 concurrently with the reduction of M1 marker, CCR7 and CD68. M2 polarization of MO in response to SP was completely blocked by NK1-R antagonist, RP67580, and PI3K inhibitor, LY294002. Furthermore, SP also showed transforming ability of M1-polarized MO induced by IFN toward M2 type with induction of CD163 expression. In summary, the results of the present study demonstrate that SP directly polarizes M2 macrophages of MO and IFN-primed M1 type and acts as a Th2 cytokine.

Cytotoxic activity of T lymphocytes is induced upon stimulation with IL-3 plus GM-CSF in animal leukemia model

Chemotherapy for leukemia has severe toxicity and bone marrow transplantation is both financially and logistically demanding. Therefore, immunotherapy is a feasible and promising approach to treat leukemia. For immunotherapy, cytotoxic T lymphocytes (CTL) against leukemic cells were induced. In BALB/c mice, leukemia was induced by N-ethyl-N′-nitrosourea (ENU). The mice were treated with recombinant IL-3 and GM-CSF – both 5μg/kg/day for four days to induce functional CTL. The IL-3+GM-CSF treatment increased total leukocyte counts, accompanied by significant increase in CTL activity, in the leukemic mice. The IL-3+GM-CSF treatment also enhanced the expression of both p40 and p35 isoforms of IL-12. Perforin and granzyme B expressions were increased in the treated group supporting the T lymphocyte-mediated cytotoxic killing of the target cells. The protein tyrosine kinase (PTK) activity was increased in leukemia but decreased after the treatment with IL-3 and GM-CSF. Interferon gamma (IFN-γ) production was decreased in leukemic condition but increased after the treatment with these colony stimulating factors. These data indicate the anti-leukemic potential of the IL-3 and GM-CSF combination therapy

IL-3 and GM-CSF in combination alters protein tyrosine kinase activity of splenic macrophages in leukemic mice.

Objectives: Murine macrophages were playing a key role against microbes and foreign particles as well as help in solid tumorigenesis in the host body. In this experiment we were trying to evaluate the protein tyrosine kinase (PTK) activity in macrophages from experimentally induced leukemic animal model.Methods: Balb/C mice were divided into four groups, two groups were challenged with N-N ethyl nitrosourea (ENU) and two groups remained without ENU challenged condition. After confirmation of leukemia induction, one normal and one ENU challenged groups were received rmIL-3 for 4 days followed by rmGM-CSF for 4 days. Disease was confirmed by histological studies of peripheral blood and bone marrow smear. PTK activity assay were done using universal protein tyrosine assay kit.Results: Protein tyrosine kinase (PTK) activity of macrophages was increased in leukemic animal significantly which were reduced after combination of IL-3 and GM-CSF treatment. IFN- ? level in blood serum were increased in leukemic mice and reduced after treatment as normal level. Conclusion: Results suggest that, in leukemia reduced macrophage number can be increased by IL-3 and GM-CSF administration in combination but PTK activity is not involved to increase the number of macrophages. PTK activity may be involved in macrophage activation process in Leukemic animal.

Dysregulation of autoantigen genes in ANCA-associated vasculitis involves alternative transcripts and new protein synthesis.

Proteinase 3 (PR3) and myeloperoxidase (MPO) are two major autoantigens in patients with vasculitis with ANCA. The genes encoding these autoantigens are abnormally expressed in peripheral granulocytes of patients with active ANCA-associated vasculitis. This study provides evidence that this transcriptional dysregulation results in a variety of mRNA processing events from the PRTN3 gene locus. In addition to elevated levels of PR3 message, leukocyte RNA from patients contained PR3 transcripts with an alternative 3' untranslated region. Furthermore, we detected usage of an alternative transcription start site within intron 1 of the PRTN3 gene locus that coincided with active disease (odds ratio, 3.3; 95% confidence interval, 1.3 to 8.4; P=0.01). This promoter may be developmentally regulated, because it was active in normal human bone marrow, multiple leukemia cell lines, MCF-7 cells, and subjects after GM-CSF treatment but not subjects with a neutrophil left shift. This transcript, which lacks exon 1 of PRTN3, encodes a 24-kD protein (p24(PR3/MBN)) with a sequence similar to that previously described for myeloblastin. Notably, PR3, p24(PR3/MBN), and MPO were synthesized in cultured neutrophils from patients with active ANCA-associated vasculitis, indicating that increased transcription results in newly synthesized autoantigens in peripheral neutrophils of patients. The synthesis of p24(PR3/MBN) seems to expand the autoantigen repertoire, because immunoblots showed that sera from patients recognized p24(PR3/MBN). These findings emphasize the importance of transcriptional dysregulation of the autoantigen in autoimmune disease.

Tumor‐associated macrophages exhibit pro‐ and anti‐inflammatory properties by which they impact on pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC) still ranking 4th in the order of fatal tumor diseases is characterized by a profound tumor stroma with high numbers of tumor-associated macrophages (TAMs). Driven by environmental factors, monocytes differentiate into M1- or M2-macrophages, the latter commonly regarded as being protumorigenic. Because a detailed analysis of TAMs in human PDAC development is still lacking, freshly isolated PDAC-derived TAMs were analyzed for their phenotype and impact on epithelial-mesenchymal-transition (EMT) of benign (H6c7) and malignant (Colo357) pancreatic ductal epithelial cells. TAMs exhibited characteristics of M1-macrophages (expression of HLA-DR, IL-1β, or TNF-α) and M2-macrophages (expression of CD163 and IL-10). In the presence of TAMs, H6c7, and Colo357 cells showed an elongated cell shape along with an increased expression of mesenchymal markers such as vimentin and reduced expression of epithelial E-cadherin. Similar to TAMs, in vitro generated M1- and M2-macrophages both mediated EMT in H6c7 and Colo357 cells. M1-macrophages acquired M2-characteristics during coculture that could be prevented by GM-CSF treatment. However, M1-macrophages still potently induced EMT in H6c7 and Colo357 cells although lacking M2-characteristics. Overall, these data demonstrate that TAMs exhibit anti- as well as proinflammatory properties that equally contribute to EMT induction in PDAC initiation and development.

Progenitor Cell Differentiation Into Dendritic Cells Requires Cytokine Mediated PKC-IRF Relationships

Dendritic Cell (DC) differentiation is a complex system involving multiple progenitors with potential to differentiate into a variety of DC subsets. Understanding the mechanisms regulating these differentiation pathways is critical to understanding how defective DCs arise in cancer. Impaired DC differentiation often results in immunosuppressive cells that either hinder immune activation in disease or promote tumor growth and metastasis. We previously established that the serine-threonine kinase Protein Kinase C β isoform II (PKCβII) is required for human DC differentiation from CD34+ progenitor cells and monocytes, and have recently found that murine bone marrow (BM) cells also need it to become fully differentiated and functional DCs. However, the molecular targets of PKCβII in this pathway remain unclear. It is well established that the transcription factors Interferon Regulatory Factors 4 and 8 (IRF4 and IRF8) are also important for DC differentiation. IRF4 is crucial for the development of conventional DCs (mediated by GM-CSF), while IRF8 is crucial for the development of plasmacytoid and CD8α+DCs (mediated by FLT3-L). We hypothesized that a relationship existed between PKCβII and IRF4/8, and investigated the effects of PKC activation on IRF4/8 expression.Using human progenitor cell lines and murine BM cells we found that PKC activation upregulated IRF4 and IRF8 expression, while PKC inhibition downregulated IRF4 and IRF8. PKC inhibition also prevented these cells from differentiating into DCs, as determined by their phenotypic markers, physical characteristics, and T-cell stimulatory activity. However, we found that in progenitor cells GM-CSF (a known PKCβII activator) decreased IRF8 expression while upregulating IRF4 expression. This led us to investigate the differential effects of GM-CSF and FLT3-L on the PKC-IRF relationship. We saw that FLT3-L treatment of murine BM cells caused an upregulation of IRF8 and stimulated DC differentiation, and that DC differentiation and IRF8 upregulation were both lost in the presence of a PKC inhibitor. Using Image Stream analysis we found that FLT3-L treatment of progenitor cells activated PKCβII and PKCα. To determine which PKC(s) mediates the FLT3-L driven upregulation of IRF8, we used PKCα knockout (KO) BM and saw that cells were still able to differentiate into DCs and IRF8 levels were still being upregulated. Thus, PKCβII is the PKC that mediates FLT3-L driven DC differentiation and IRF8 upregulation. To determine what molecules could be acting downstream of PKCβII in regulating IRF4/8, we again used Image Stream analysis and visualized STAT3 and STAT5 translocation into the nucleus. Using murine BM cells we found that STAT3 and IRF8 nuclear localization increased with FLT3-L treatment, while GM-CSF treatment caused increased STAT5 and IRF4 nuclear localization. When looking at human monocytes and the human monocytic progenitor cell lineTHP-1 we saw similar effects: GM-CSF treatment increased STAT5 and IRF4 nuclear localization, while pan-PKC inhibition decreased basal STAT5 and IRF4 nuclear localization. Interestingly, these human monocytes and THP-1 cells had lower nuclear levels of STAT3 and IRF8 following FLT3-L treatment – possibly because these cells are already somewhat committed to the monocyte-derived conventional DC pathway. However, in murine early progenitor cells, after 15 minutes of PKC activation we saw increased STAT3 activation, indicating that PKC-regulated STAT3 activation is playing a role earlier in the differentiation process. To find the progenitor cells immediately effected by PKCβII activation, we used IRF8-eGFP murine BM and saw that PKC activation caused induced IRF8 expression as early as in the multi-potent progenitor cells (MPP2 and MPP3), and this upregulation continued to increase as cells differentiated to CD11b+progenitor cells and GMP.These studies indicate that PKCβII is activated in progenitor cells by either FLT3-L or GM-CSF, causing an upregulation of IRF8 or IRF4, respectively. PKCβII may be acting through STAT5 and STAT3 to induce IRF4 and IRF8, depending on the cytokine treatment. By having a better understanding of how PKCβII regulates the expression of these transcription factors, which are required for DC differentiation, we can manipulate the PKCβII-IRF relationship to drive or impair DC differentiation in pathological settings, and may improve DC-vaccine development. Disclosures:No relevant conflicts of interest to declare.

Human Airway Eosinophils Exhibit Preferential Reduction in STAT Signaling Capacity and Increased CISH Expression

Allergic asthma, a chronic respiratory disorder marked by inflammation and recurrent airflow obstruction, is associated with elevated levels of IL-5 family cytokines and elevated numbers of eosinophils (EOS). IL-5 family cytokines elongate peripheral blood EOS (EOS(PB)) viability, recruit EOS(PB) to the airways, and, at higher concentrations, induce degranulation and reactive oxygen species generation. Although airway EOS (EOS(A)) remain signal ready in that GM-CSF treatment induces degranulation, treatment of EOS(A) with IL-5 family cytokines no longer confers a survival advantage. Because the IL-5 family receptors have common signaling capacity, but are uncoupled from EOS(A) survival, whereas other IL-5 family induced endpoints remain functional, we tested the hypothesis that EOS(A) possess a JAK/STAT-specific regulatory mechanism (because JAK/STAT signaling is critical to EOS survival). We found that IL-5 family-induced STAT3 and STAT5 phosphorylation is attenuated in EOS(A) relative to blood EOS from airway allergen-challenged donors. However, IL-5 family-induced ERK1/2 phosphorylation is not altered between EOS(A) and EOS from airway allergen-challenged donors. These observations suggest EOS(A) possess a regulatory mechanism for suppressing STAT signaling distinct from ERK1/2 activation. Furthermore, we found, in EOS(PB), IL-5 family cytokines induce members of the suppressors of cytokine signaling (SOCS) genes, CISH and SOCS1. Additionally, following allergen challenge, EOS(A) express significantly more CISH and SOCS1 mRNA and CISH protein than EOS(PB) counterparts. In EOS(PB), long-term pretreatment with IL-5 family cytokines, to varying degrees, attenuates IL-5 family-induced STAT5 phosphorylation. These data support a model in which IL-5 family cytokines trigger a selective downregulation mechanism in EOS(A) for JAK/STAT pathways.

Depletion of Alveolar Macrophages during Influenza Infection Facilitates Bacterial Superinfections

Viruses such as influenza suppress host immune function by a variety of methods. This may result in significant morbidity through several pathways, including facilitation of secondary bacterial pneumonia from pathogens such as Streptococcus pneumoniae. PKH26-phagocytic cell labeling dye was administered intranasally to label resident alveolar macrophages (AMs) in a well-established murine model before influenza infection to determine turnover kinetics during the course of infection. More than 90% of resident AMs were lost in the first week after influenza, whereas the remaining cells had a necrotic phenotype. To establish the impact of this innate immune defect, influenza-infected mice were challenged with S. pneumoniae. Early AM-mediated bacterial clearance was significantly impaired in influenza-infected mice: ~50% of the initial bacterial inoculum could be harvested from the alveolar airspace 3 h later. In mock-infected mice, by contrast, >95% of inocula up to 50-fold higher was efficiently cleared. Coinfection during the AM depletion phase caused significant body weight loss and mortality. Two weeks after influenza, the AM population was fully replenished with successful re-establishment of early innate host protection. Local GM-CSF treatment partially restored the impaired early bacterial clearance with efficient protection against secondary pneumococcal pneumonia. We conclude that resident AM depletion occurs during influenza infection. Among other potential effects, this establishes a niche for secondary pneumococcal infection by altering early cellular innate immunity in the lungs, resulting in pneumococcal outgrowth and lethal pneumonia. This novel mechanism will inform development of novel therapeutic approaches to restore lung innate immunity against bacterial superinfections.