Hong R. Cho

IL-33 Priming Regulates Multiple Steps of the Neutrophil-Mediated Anti-Candida albicans Response by Modulating TLR and Dectin-1 Signals

IL-33 is known to play an important role in Th2 immunity. In this study, we investigated the effect of IL-33 pretreatment on anti-fungal response using an acute Candida albicans peritoneal infection model. IL-33 pretreatment induced a rapid fungal clearance and markedly reduced the C. albicans infection-associated mortality. The priming effect of IL-33 occurred during multiple steps of the neutrophil-mediated anti-fungal response. First, the anti-fungal effect occurred due to the rapid and massive recruitment of neutrophils to the site of infection as a result of the release of CXCR2 chemokines by peritoneal macrophages and by reversal of the TLR-induced reduction of CXCR2 expression in neutrophils during IL-33 priming. Second, conditioning of neutrophils by IL-33 activated the TLR and dectin-1 signaling pathways, leading to the upregulation of complement receptor 3 expression induced by C. albicans. Upregulated CR3 in turn increased the phagocytosis of opsonized C. albicans and resulted in the production of high levels of reactive oxygen species and the subsequent enhanced killing activity of neutrophils. Taken together, our results suggest that IL-33 can regulate the anti-fungal activity of neutrophils by collaborative modulation of the signaling pathways of different classes of innate immune receptors.

TLR2 Signaling in Tubular Epithelial Cells Regulates NK Cell Recruitment in Kidney Ischemia–Reperfusion Injury

Damage-associated molecular patterns released from damaged kidney cells initiate postischemic inflammation, an essential step in the progression of kidney ischemia–reperfusion injury (IRI). However, the mechanism that coordinates this highly specific process in ischemic kidneys remains to be clarified. Previously, we demonstrated that CD137 from NK cells specifically stimulates CD137 ligand (CD137L) on tubular epithelial cells (TECs) such that TECs produced the high CXCR2 chemokine levels required for neutrophil chemotaxis. We report in the present study that endogenous TLR2 ligands released from ischemic TECs induce CCR5 chemokine expression, which is critical to promoting NK cell recruitment. By implanting CD137L−/− TECs into the kidney capsule of TLR2−/− mice, we further showed that TLR2-mediated NK cell recruitment is an uncoupled event that can occur independently of CD137L signaling in TECs, which is responsible for recruiting neutrophils. Therefore, our findings identify TECs as both a target for kidney damage and also as a master regulator that actively modulates stepwise signaling, leading to the initiation and amplification of acute sterile inflammation that inflicts kidney IRI. Being clinically important, the signaling pathway of innate receptors in epithelial cells may therefore be a good target to block acute sterile inflammation resulting from tissue damage, including kidney IRI.

Interleukin-33: a mediator of inflammation targeting hematopoietic stem and progenitor cells and their progenies.

Inflammation is defined as a physiological response initiated by a variety of conditions that cause insult to the body, such as infection and tissue injury. Inflammation is triggered by specialized receptors in the innate immune system, which recognize microbial components known as pathogen-associated molecular patterns or endogenous signals produced by damaged cells (damage-associated molecular patterns). IL-33 is a cytokine that is released predominantly at the epithelial barrier when it is exposed to pathogens, allergens, or injury-inducing stimuli. IL-33 target cells are various, ranging from hematopoietic stem and progenitor cells (HSPCs) and essentially all types of their progeny to many non-hematopoietic cells. The pleiotropic actions of IL-33 suggest that IL-33 is involved in every phase of the inflammatory process. In this review, we discuss recent advances in the understanding of how IL-33 orchestrates inflammatory responses by regulating HSPCs and innate immune cells.

IL-33–Induced Hematopoietic Stem and Progenitor Cell Mobilization Depends upon CCR2

IL-33 has been implicated in the pathogenesis of asthma, atopic allergy, anaphylaxis, and other inflammatory diseases by promoting the production of proinflammatory cytokines and chemokines or Th2 immune responses. In this study, we analyzed the in vivo effect of IL-33 administration. IL-33 markedly promoted myelopoiesis in the bone marrow and myeloid cell emigration. Concomitantly, IL-33 induced hematopoietic stem and progenitor cell (HSPC) mobilization and extramedullary hematopoiesis. HSPC mobilization was mediated mainly through increased levels of CCL7 produced by vascular endothelial cells in response to IL-33. In vivo treatment of IL-33 rapidly induced phosphorylation of ERK, JNK, and p38, and inhibition of these signaling molecules completely blocked the production of CCL7 induced by IL-33. Consistently, inhibitor of CCR2 markedly reduced IL-33–mediated HSPC mobilization in vivo and migration of HSPCs in response to CCL7 in vitro. IL-33–mobilized HSPCs were capable of homing to, and of long-term reconstitution in, the bone marrow of irradiated recipients. Immune cells derived from these recipients had normal antifungal activity. The ability of IL-33 to promote migration of HSPCs and myeloid cells into the periphery and to regulate their antifungal activity represents a previously unrecognized role of IL-33 in innate immunity. These properties of IL-33 have clinical implications in hematopoietic stem cell transplantation.

IL-33 Enhances Host Tolerance to Candida albicans Kidney Infections through Induction of IL-13 Production by CD4+ T Cells

Susceptibility to systemic Candida albicans infection is determined by immune resistance, as well as by the ability to control Candida-induced immunopathologies. We showed previously that exogenous IL-33 can increase resistance to peritoneal C. albicans infection by regulating multiple steps of the neutrophil anti-Candida response. In this study, using a mouse model of systemic candidiasis, we observed that IL-33 administration limited fungal burden and inflammation and increased survival. In kidneys, IL-33 seemed to directly act on neutrophils and CD4+ T cells: IL-33 administration enhanced fungal clearance by increasing neutrophil phagocytic activity without which Candida proliferation was uncontrollable. In contrast, IL-33 stimulated CD4+ T cells to produce IL-13, which, in turn, drove the polarization of macrophages toward the M2 type. Furthermore, the absence of IL-13 abolished IL-33–mediated polarization of M2 macrophages and renal functional recovery. In addition, IL-33 and IL-13 acted synergistically to increase M2 macrophage polarization and its phagocytic activity. Overall, this study identifies IL-33 as a cytokine that is able to induce resistance and tolerance and suggests that targeting resistance and tolerance simultaneously with therapeutic IL-33 may benefit patients with systemic candidiasis.

Intratumorally Establishing Type 2 Innate Lymphoid Cells Blocks Tumor Growth

A long-standing question in the field of tumor immunotherapy is how Th2 cytokines block tumor growth. Their antitumor effects are particularly prominent when they are secreted continuously in tumors, suggesting that Th2 cytokines may create a tumor microenvironment unfavorable for tumor growth independently of adaptive immunity. In this study, we show that local production of IL-33 establishes a high number of type 2 innate lymphoid cells (ILC2s) with potent antitumor activity. IL-33 promotes secretion of a massive amount of CXCR2 ligands from ILC2s but creates a tumor microenvironment where tumor cells express CXCR2 through a dysfunctional angiogenesis/hypoxia/reactive oxygen species axis. These two signaling events converge to reinforce tumor cell–specific apoptosis through CXCR2. Our results identify a previously unrecognized antitumor therapeutic pathway wherein ILC2s play a central role.

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease.

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.

A novel mouse PKCδ splice variant, PKCδIX, inhibits etoposide-induced apoptosis.

Protein kinase C (PKC) δ plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKCδ generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKCδ isoform named PKCδIX (Genebank Accession No. HQ840432). PKCδIX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKCδ. PKCδIX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKCδIX provided a possibility that this PKCδ isozyme functions as a novel dominant-negative form for PKCδ due to its lack of the ATP-binding domain that is required for the kinase activity of PKCδ. Indeed, overexpression of PKCδIX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKCδIX protein could competitively inhibit the kinase activity of PKCδ. We conclude that PKCδIX can function as a natural dominant-negative inhibitor of PKCδin vivo.

A novel method for procuring a large quantity of mature murine eosinophils in vivo.

Eosinophils are rare hematopietic cells that normally constitute only 1~3% of peripheral blood leukocytes. It would be of help for the purpose of research to obtain a large quantity of eosinophils. In this study, we wanted to develop a novel strategy to induce massive expansion of murine eosinophils in vivo, based on the observation showing that treatment of IL-33 induces eosinophilia in mice. We generated an EL-4 lymphoma cell line (herein named EL-4-IL-33) that was engineered to secrete an active form of IL-33. We found that Siglec-F(+) granulocyte numbers increased by 1850-fold in the peritoneal cavity 10 days after inoculation with 1×10⁷ EL-4-IL-33 cells. This number corresponds to 74-fold increase, as compared with the number of Siglec-F(+) granulocytes in mice that received wild-type EL-4 cells. Siglec-F(+) granulocytes expanded by IL-33 had the circular nucleus and expressed eosinophil-specific genes. They also showed some functional characteristics of eosinophils in that they had the ability to respond to IL-5 for survival and eotaxin-1 for chemoattaxis and to produce bioactive eosinophil peroxidase, suggesting that these cells are genuine eosinophils. Our results indicate that sustained secretion of IL-33 by lymphoma cells in the peritoneal cavity is highly effective in increasing peritoneal eosinophil numbers. Therefore, our simple method to obtain eosinphils on a large scale might be of value for eosinophil studies.

Anti-CD137 suppresses tumor growth by blocking reverse signaling by CD137 ligand

CD137 (4-1BB) is a T-cell costimulatory molecule, and agonstic CD137 antibodies are currently being evaluated in the clinic as cancer immunotherapy. Recently, it was found that CD137-/- mice or mice injected with agonistic anti-CD137 antibodies exhibit heightened antitumor responses, contrary to expectations based on other knowledge of CD137 function. Here, we report findings related to reverse signaling by CD137 ligand (CD137L) in antigen-presenting dendritic cells (DC) in tumors that address these paradoxical results. Specifically, CD137L suppressed intratumoral differentiation of IL12-producing CD103+ DC and type 1 tumor-associated macrophages (TAM). Differentiation of these cell types is important because they are required to generate IFNγ-producing CD8+ cytotoxic T lymphocytes (Tc1). Notably, CD137L blockade increased levels of IL12 and IFNγ, which promoted intratumoral differentiation of IFNγ-producing Tc1, IL12-producing CD103+ DC, and type 1 TAM within tumors. Our results offer an explanation for the paradoxical effects of CD137 blockade, based on differential immunomodulatory effects of CD137 signaling and reverse signaling in T cells and DC, respectively. Further, they show how CD137L blockade can seed a forward-feedback loop for activation of CD103+ DC/type 1 TAM and Tc1 that can create a self-perpetuating cycle of highly effective immunosurveillance. Cancer Res; 77(21); 5989-6000. ©2017 AACR.