Yaron Carmi

IL-1α and IL-1β Recruit Different Myeloid Cells and Promote Different Stages of Sterile Inflammation

The immune system has evolved to protect the host from invading pathogens and to maintain tissue homeostasis. Although the inflammatory process involving pathogens is well documented, the intrinsic compounds that initiate sterile inflammation and how its progression is mediated are still not clear. Because tissue injury is usually associated with ischemia and the accompanied hypoxia, the microenvironment of various pathologies involves anaerobic metabolites and products of necrotic cells. In the current study, we assessed in a comparative manner the role of IL-1α and IL-1β in the initiation and propagation of sterile inflammation induced by products of hypoxic cells. We found that following hypoxia, the precursor form of IL-1α, and not IL-1β, is upregulated and subsequently released from dying cells. Using an inflammation-monitoring system consisting of Matrigel mixed with supernatants of hypoxic cells, we noted accumulation of IL-1α in the initial phase, which correlated with the infiltration of neutrophils, and the expression of IL-1β correlated with later migration of macrophages. In addition, we were able to show that IL-1 molecules from cells transfected with either precursor IL-1α or mature IL-1β can recruit neutrophils or macrophages, respectively. Taken together, these data suggest that IL-1α, released from dying cells, initiates sterile inflammation by inducing recruitment of neutrophils, whereas IL-1β promotes the recruitment and retention of macrophages. Overall, our data provide new insight into the biology of IL-1 molecules as well as on the regulation of sterile inflammation.

Differential release of chromatin-bound IL-1α discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation

IL-1α, like IL-1β, possesses multiple inflammatory and immune properties. However, unlike IL-1β, the cytokine is present intracellularly in healthy tissues and is not actively secreted. Rather, IL-1α translocates to the nucleus and participates in transcription. Here we show that intracellular IL-1α is a chromatin-associated cytokine and highly dynamic in the nucleus of living cells. During apoptosis, IL-1α concentrates in dense nuclear foci, which markedly reduces its mobile nature. In apoptotic cells, IL-1α is retained within the chromatin fraction and is not released along with the cytoplasmic contents. To simulate the in vivo inflammatory response to cells undergoing different mechanisms of death, lysates of cells were embedded in Matrigel plugs and implanted into mice. Lysates from cells undergoing necrosis recruited cells of the myeloid lineage into the Matrigel, whereas lysates of necrotic cells lacking IL-1α failed to recruit an infiltrate. In contrast, lysates of cells undergoing apoptotic death were inactive. Cells infiltrating the Matrigel were due to low concentrations (20–50 pg) of the IL-1α precursor containing the receptor interacting C-terminal, whereas the N-terminal propiece containing the nuclear localization site failed to do so. When normal keratinocytes were subjected to hypoxia, the constitutive IL-1α precursor was released into the supernatant. Thus, after an ischemic event, the IL-1α precursor is released by hypoxic cells and incites an inflammatory response by recruiting myeloid cells into the area. Tissues surrounding the necrotic site also sustain damage from the myeloid cells. Nuclear trafficking and differential release during necrosis vs. apoptosis demonstrate that inflammation by IL-1α is tightly controlled.

The Role of Macrophage-Derived IL-1 in Induction and Maintenance of Angiogenesis

Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

An interactive reference framework for modeling a dynamic immune system

Single-cell measurements map immunity Multiple characteristics of individual cells define cell types and their physiological states. Spitzer et al. quantitated the abundance of 39 different cell surface proteins or transcription factors on individual cells of the mouse immune system. They used these measurements to create a map that clustered similar individual cells into groups corresponding to cell type and function. Their extensible experimental platform will allow the inclusion of other data types and data from independent laboratories. Science, this issue 10.1126/science.1259425 Cytometry meets mass spectrometry to create a functional map of the immune system. INTRODUCTION Immune cells constitute an interacting hierarchy that coordinates its activities according to genetic and environmental contexts. This systemically mobile network of cells results in emergent properties that are derived from dynamic cellular interactions. Unlike many solid tissues, where cells of given functions are localized into substructures that can be readily defined, the distribution of phenotypically similar immune cells into various organs complicates discerning any modest differences between them. Over decades of investigation into immune functions during health and disease, research has necessarily focused on understanding the individual cell types within the immune system, and, more recently, toward identifying interacting cells and the messengers they use to communicate. RATIONALE Methods of single-cell analysis, such as flow cytometry, have led the effort to enumerate and quantitatively characterize immune cell populations. As research has accelerated, our understanding of immune organization has surpassed the technical limitations of fluorescence-based flow cytometry. With the advent of mass cytometry, which enables measuring significantly more features of individual cells, most known immune cell types can now be identified from within a single experiment. Leveraging this capability, we set out to initiate an immune system reference framework to provide a working definition of immune organization and enable the integration of new data sets. RESULTS To build a reference framework from mass cytometry data, we developed a novel algorithm to transform the single-cell data into intuitive maps. These Scaffold maps provide a data-driven interpretation of immune organization while also integrating conventional immune cell populations as landmarks to orient the user. By applying Scaffold maps to data from the bone marrow of wild-type C57BL/6 mice, the method reconstructed the organization within this complex developmental organ. Using this sample as a reference point, the unique organization of immune cells within various organs across the body was revealed. The maps recapitulated canonical cellular phenotypes while revealing reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune structure, permitted direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. CONCLUSION This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology. Beyond providing an analytical framework to understand immune organization from the unified data set generated here, the approaches we describe can serve as a data repository for collating experimental data from the research community, including gene expression and mutational analysis. Efforts that characterize cellular behavior in this open-source approach will continue to improve upon the initiating reference presented here to reveal the inherent structure in biological networks of immunity for clinical benefit. Building a dynamic immune system reference framework. By combining mass cytometry with the Scaffold maps algorithm, the cellular organization of any complex sample can be transformed into an intuitive and interactive map for further analysis. By first choosing one foundational sample as a reference (i.e., the bone marrow of wild-type mice), the effects of any perturbation can be readily identified in this framework. Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

Interleukin-1β regulates fat-liver crosstalk in obesity by auto-paracrine modulation of adipose tissue inflammation and expandability.

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.

The Role of IL-1β in the Early Tumor Cell–Induced Angiogenic Response

In this study, we assessed the involvement of IL-1β in early angiogenic responses induced by malignant cells using Matrigel plugs supplemented with B16 melanoma cells. We found that during the angiogenic response, IL-1β and vascular endothelial growth factor (VEGF) interact in a newly described autoinduction circuit, in which each of these cytokines induces the other. The IL-1β and VEGF circuit acts through interactions between bone marrow–derived VEGF receptor 1+/IL-1R1+ immature myeloid cells and tissue endothelial cells. Myeloid cells produce IL-1β and additional proinflammatory cytokines, which subsequently activate endothelial cells to produce VEGF and other proangiogenic factors and provide the inflammatory microenvironment for angiogenesis and tumor progression. These mechanisms were also observed in a nontumor early angiogenic response elicited in Matrigel plugs by either rIL-1β or recombinant VEGF. We have shown that IL-1β inhibition stably reduces tumor growth by limiting inflammation and inducing the maturation of immature myeloid cells into M1 macrophages. In sharp contrast, only transient inhibition of tumor growth was observed after VEGF neutralization, followed by tumor recurrence mediated by rebound angiogenesis. This occurs via the reprogramming of VEGF receptor 1+/IL-1R1+ cells to express hypoxia inducible factor-1α, VEGF, and other angiogenic factors, thereby directly supporting proliferation of endothelial cells and blood vessel formation in a paracrine manner. We suggest using IL-1β inhibition as an effective antitumor therapy and are currently optimizing the conditions for its application in the clinic.

The role IL-1 in tumor-mediated angiogenesis

Tumor angiogenesis is one of the hallmarks of tumor progression and is essential for invasiveness and metastasis. Myeloid inflammatory cells, such as immature myeloid precursor cells, also termed myeloid-derived suppressor cells (MDSCs), neutrophils, and monocytes/macrophages, are recruited to the tumor microenvironment by factors released by the malignant cells that are subsequently “educated” in situ to acquire a pro-invasive, pro-angiogenic, and immunosuppressive phenotype. The proximity of myeloid cells to endothelial cells (ECs) lining blood vessels suggests that they play an important role in the angiogenic response, possibly by secreting a network of cytokines/chemokines and inflammatory mediators, as well as via activation of ECs for proliferation and secretion of pro-angiogenic factors. Interleukin-1 (IL-1) is an “alarm,” upstream, pro-inflammatory cytokine that is generated primarily by myeloid cells. IL-1 initiates and propagates inflammation, mainly by inducing a local cytokine network and enhancing inflammatory cell infiltration to affected sites and by augmenting adhesion molecule expression on ECs and leukocytes. Pro-inflammatory mediators were recently shown to play an important role in tumor-mediated angiogenesis and blocking their function may suppress tumor progression. In this review, we summarize the interactions between IL-1 and other pro-angiogenic factors during normal and pathological conditions. In addition, the feasibility of IL-1 neutralization approaches for anti-cancer therapy is discussed.

Microenvironment-Derived IL-1 and IL-17 Interact in the Control of Lung Metastasis

Inflammatory cytokines modulate immune responses in the tumor microenvironment during progression/metastasis. In this study, we have assessed the role of IL-1 and IL-17 in the control of antitumor immunity versus progression in a model of experimental lung metastasis, using 3LL and B16 epithelial tumor cells. The absence of IL-1 signaling or its excess in the lung microenvironment (in IL-1β and IL-1R antagonist knockout [KO] mice, respectively) resulted in a poor prognosis and reduced T cell activity, compared with WT mice. In IL-1β KO mice, enhanced T regulatory cell development/function, due to a favorable in situ cytokine network and impairment in APC maturation, resulted in suppressed antitumor immunity, whereas in IL-1R antagonist KO mice, enhanced accumulation and activity of myeloid-derived suppressor cells were found. Reduced tumor progression along with improved T cell function was found in IL-17 KO mice, compared with WT mice. In the microenvironment of lung tumors, IL-1 induces IL-17 through recruitment of γ/δ T cells and their activation for IL-17 production, with no involvement of Th17 cells. These interactions were specific to the microenvironment of lung tumors, as in intrafootpad tumors in IL-1/IL-17 KO mice, different patterns of invasiveness were observed and no IL-17 could be locally detected. The results highlight the critical and unique role of IL-1, and cytokines induced by it such as IL-17, in determining the balance between inflammation and antitumor immunity in specific tumor microenvironments. Also, we suggest that intervention in IL-1/IL-17 production could be therapeutically used to tilt this balance toward enhanced antitumor immunity.

p53 Status in Stromal Fibroblasts Modulates Tumor Growth in an SDF1-Dependent Manner

The p53 tumor suppressor exerts a variety of cell-autonomous effects that are aimed to thwart tumor development. In addition, however, there is growing evidence for cell nonautonomous tumor suppressor effects of p53. In the present study, we investigated the impact of stromal p53 on tumor growth. Specifically, we found that ablation of p53 in fibroblasts enabled them to promote more efficiently the growth of tumors initiated by PC3 prostate cancer-derived cells. This stimulatory effect was dependent on the increased expression of the chemokine SDF-1 in the p53-deficient fibroblasts. Notably, fibroblasts harboring mutant p53 protein were more effective than p53-null fibroblasts in promoting tumor growth. The presence of either p53-null or p53-mutant fibroblasts led also to a markedly elevated rate of metastatic spread of the PC3 tumors. These findings implicate p53 in a cell nonautonomous tumor suppressor role within stromal fibroblasts, through suppressing the production of tumor stimulatory factors by these cells. Moreover, expression of mutant p53 by tumor stroma fibroblasts might exert a gain of function effect, further accelerating tumor development.

Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity

Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.