Eosinophils oxidize damage-associated molecular pattern molecules derived from stressed cells.

Eosinophils (Eos) are found at increased numbers within necrotic areas of tumors. We show that necrotic material from cell lysates containing damage-associated molecular pattern molecules induce eosinophil degranulation (release of major basic protein and eosinophil peroxidase) and enhance their oxidative burst while the stimulatory capacity of cell lysates is significantly diminished following oxidation. High mobility group box 1 (HMGB1), a prototypic damage-associated molecular pattern molecule, released following necrosis but not apoptosis, induced a similar effect on Eos. Additionally, we demonstrate that HMGB1 enhances eosinophil survival and acts as a chemoattractant. Consistently, we show that Eos express an HMGB1 receptor, the receptor for advanced glycation end product, and that anti-receptor for advanced glycation end product could diminish the HMGB1-mediated effects. Of all tested biologic activities, Eos respond most sensitively to the presence of necrotic material including HMGB1 with generation of peroxide. We postulate that Eos "sense" necrotic cell death, migrating to and responding to areas of tissue injury/necrosis. Oxidation of cell lysates reduces their biologic activity when compared with native lysates. We postulate that eosinophil-associated modulation of immunity within tumor and other damaged tissues may be primarily by promoting oxidative degradation of necrotic material. Novel therapeutic strategies may be considered by advancing oxidative denaturation of released necrotic material using Eos or other aerobic strategies.

Blocking drug-induced autophagy with chloroquine in HCT-116 colon cancer cells enhances DC maturation and T cell responses induced by tumor cell lysate.

Autophagy is an important mechanism for tumor escape, allowing tumor cells to recover from the damage induced by chemotherapy, radiation therapy, and immunotherapy and contributing to the development of resistance. The pharmacological inhibition of autophagy contributes to increase the efficacy of antineoplastic agents. Exposing tumor cells to low concentrations of select autophagy-inducing antineoplastic agents increases their immunogenicity and enhances their ability to stimulate dendritic cell (DC) maturation. We tested whether the application of an autophagy-inhibiting agent, chloroquine (CQ), in combination with low concentrations of 5-fluorouracil (5-FU) increases the ability of tumor cells to induce DC maturation. DCs sensitized with the lysate of HCT-116 cells previously exposed to such a combination enhanced the DC maturation/activation ability. These matured DCs also increased the allogeneic responsiveness of both CD4+ and CD8+ T cells, which showed a greater proliferative response than those from DCs sensitized with control lysates. The T cells expanded in such cocultures were CD69+ and PD-1- and produced higher levels of IFN-γ and lower levels of IL-10, consistent with the preferential activation of Th1 cells. Cocultures of autologous DCs and lymphocytes improved the generation of cytotoxic T lymphocytes, as assessed by the expression of CD107a, perforin, and granzyme B. The drug combination increased the expression of genes related to the CEACAM family (BECN1, ATGs, MAPLC3B, ULK1, SQSTM1) and tumor suppressors (PCBP1). Furthermore, the decreased expression of genes related to metastasis and tumor progression (BNIP3, BNIP3L, FOSL2, HES1, LAMB3, LOXL2, NDRG1, P4HA1, PIK3R2) was noted. The combination of 5-FU and CQ increases the ability of tumor cells to drive DC maturation and enhances the ability of DCs to stimulate T cell responses.

Eosinophils induce DC maturation, regulating immunity.

There are increased eosinophils in tumors, and they are generally associated with a good prognosis, whereas their presence in rejecting allografts is largely seen as a harbinger of poor outcome. The biologic role of eosinophils in their pathogenesis is more poorly understood than in allergy and asthma. Myeloid conventional dendritic cells (DCs) and conversely, plasmacytoid DCs are similarly associated with a good prognosis in cancer patients. We hypothesize that eosinophils, similar to NK cells, could mature DCs, and that could be responsible for regulating immunity in the setting of necrosis-associated chronic inflammation as occurs in cancer and transplant rejection. We have demonstrated that CpG DNA promotes eosinophil-induced DC maturation. As such, a greater linkage than had previously been considered between innate immune cells such as eosinophils and the adaptive immune response can be considered. Granulocytes were isolated from normal human whole blood by density gradient centrifugation followed by ammonium chloride-potassium lysis of the remaining red cells. Eosinophils were negatively separated using magnetic beads. Immature DCs were generated from CD-14 positively separated monocytes, which were cultured for 6 days with GM-CSF and IL-4. CpG ODN 2395 (CpG-C) as a pathogen-associated molecular pattern surrogate was used to induce eosinophil-based DC maturation. Transwells were used to assess cell-cell interaction between eosinophils and DCs. Eosinophil survival was assessed by flow cytometry; cells, which did not stain with Sytox-Orange, were considered viable. In the presence of CpG-C, eosinophils induced DC maturation. Similar results were obtained when eosinophils were pretreated with CpG for 4 h, washed, and cocultured afterwards with DCs. Eosinophil-induced maturation of DCs directly correlated with the eosinophil:DC ratio. Transwell studies showed that the direct cell-cell interaction between eosinophils and DCs enhances maturation. CpGs did not adversely affect eosinophil survival; thus, we could exclude the possibility that DC maturation was caused by sensing eosinophil cell death. While eosinophil-derived neurotoxin did not contribute to the described effect, DCs took up and internalized major basic protein (MBP), which was released from CpG-stimulated eosinophils, revealed by confocal imaging and flow cytometry. Thus, the DC maturational-inducing effect of eosinophils may be a result of released MBP from eosinophils. CpG-activated eosinophils mature conventional DCs. The role of viral or bacterial products or potentially, host-derived DNA as eosinophil activators with consequent DC maturation should be considered in more detail in the inflammatory settings in which eosinophils have been observed.

Immunotherapy for cancer: promoting innate immunity.

Development of tumor over many years leads to reciprocal alterations in the host immune response and the tumor, enabling tumor growth seemingly paradoxically in the setting of necrosis and inflammation. Innate immune cells, granulocytes - neutrophils, eosinophils, basophils - and mast cells belong to the first line of defense sensing pathogen and damage associated molecular pattern (PAMPs, DAMPs) signals, initiating and modulating the subsequent inflammatory response. Nontheless, the prevailing contemporary strategies of immunotherapy for cancer have focused on the second line of the immune response, the adaptive immune response. We have determined that most highly evolved tumors in adults undergo necrosis, releasing DAMPs, promoting reactive angiogenesis, stromagenesis and reparative epithelial proliferation of the tumor cell. Means to aerobically eliminate such DAMPs by peroxidases released by innate immune effectors allows us to consider novel strategies for limiting tumor progression. Summarized here is our current understanding of acute and chronic inflammation and its impact on tumor development, the pathophysiology of immunity in cancer, and the influence of granulocytes and mast cells in this setting.