Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.

HMGB1 promotes CXCL12-dependent egress of murine B cells from Peyer's patches in homeostasis.

High mobility group box-1 protein (HMGB1) is an alarmin that, once released, promotes inflammatory responses, alone and as a complex with the chemokine CXCL12. Here, we report that the HMGB1-CXCL12 complex plays an essential role also in homeostasis by controlling the migration of B lymphocytes. We show that extracellular HMGB1 is critical for the CXCL12-dependent egress of B cells from the Peyer's patches (PP). This promigratory function of the complex was restricted to the PPs, since HMGB1 was not required for B-cell migratory processes in other locations. Accordingly, we detected higher constitutive levels of the HMGB1-CXCL12 complex in PPs than in other lymphoid organs. HMGB1-CXCL12 in vivo inhibition was associated with a reduced basal IgA production in the gut. Collectively, our results demonstrate a role for the HMGB1-CXCL12 complex in orchestrating B-cell trafficking in homeostasis, and provide a novel target to control lymphocyte migration in mucosal immunity.

Nucleosomes effectively shield DNA from radiation damage in living cells.

Eukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. Here, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. We combined sequence-based methods (ATAC-seq and BLISS) to determine the position of both nucleosomes and double strand breaks (DSBs) in the genome of nucleosome-rich malignant mesothelioma cells, and of the same cells partially depleted of nucleosomes. The results were replicated in the human MCF-7 breast carcinoma cell line. We found that, for each genomic sequence, the probability of DSB formation is directly proportional to the fraction of time it is nucleosome-free; DSBs accumulate distal from the nucleosome dyad axis. Nucleosome free regions and promoters of actively transcribed genes are more sensitive to DSB formation, and consequently to mutation. We argue that this may be true for a variety of chemical and physical DNA damaging agents.

Redox modifications of cysteine residues regulate the cytokine activity of HMGB1.

BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein with extracellular inflammatory cytokine activity. It is passively released during cell death and secreted by activated cells of many lineages. HMGB1 contains three conserved redox-sensitive cysteine residues: cysteines in position 23 and 45 (C23 and C45) can form an intramolecular disulfide bond, whereas C106 is unpaired and is essential for the interaction with Toll-Like Receptor (TLR) 4. However, a comprehensive characterization of the dynamic redox states of each cysteine residue and of their impacts on innate immune responses is lacking. METHODS: Primary human macrophages or murine macrophage-like RAW 264.7 cells were activated in cell cultures by redox-modified or point-mutated (C45A) recombinant HMGB1 preparations or by lipopolysaccharide (E. coli.0111: B4). Cellular phosphorylated NF-κB p65 subunit and subsequent TNF-α release were quantified by commercial enzyme-linked immunosorbent assays. RESULTS: Cell cultures with primary human macrophages and RAW 264.7 cells demonstrated that fully reduced HMGB1 with all three cysteines expressing thiol side chains failed to generate phosphorylated NF-КB p65 subunit or TNF-α. Mild oxidation forming a C23-C45 disulfide bond, while leaving C106 with a thiol group, was required for HMGB1 to induce phosphorylated NF-КB p65 subunit and TNF-α production. The importance of a C23-C45 disulfide bond was confirmed by mutation of C45 to C45A HMGB1, which abolished the ability for cytokine induction. Further oxidation of the disulfide isoform also inactivated HMGB1. CONCLUSIONS: These results reveal critical post-translational redox mechanisms that control the proinflammatory activity of HMGB1 and its inactivation during inflammation.

CXCL10 levels at hospital admission predict COVID-19 outcome: hierarchical assessment of 53 putative inflammatory biomarkers in an observational study.

BACKGROUND: Host inflammation contributes to determine whether SARS-CoV-2 infection causes mild or life-threatening disease. Tools are needed for early risk assessment. METHODS: We studied in 111 COVID-19 patients prospectively followed at a single reference Hospital fifty-three potential biomarkers including alarmins, cytokines, adipocytokines and growth factors, humoral innate immune and neuroendocrine molecules and regulators of iron metabolism. Biomarkers at hospital admission together with age, degree of hypoxia, neutrophil to lymphocyte ratio (NLR), lactate dehydrogenase (LDH), C-reactive protein (CRP) and creatinine were analysed within a data-driven approach to classify patients with respect to survival and ICU outcomes. Classification and regression tree (CART) models were used to identify prognostic biomarkers. RESULTS: Among the fifty-three potential biomarkers, the classification tree analysis selected CXCL10 at hospital admission, in combination with NLR and time from onset, as the best predictor of ICU transfer (AUC [95% CI] = 0.8374 [0.6233-0.8435]), while it was selected alone to predict death (AUC [95% CI] = 0.7334 [0.7547-0.9201]). CXCL10 concentration abated in COVID-19 survivors after healing and discharge from the hospital. CONCLUSIONS: CXCL10 results from a data-driven analysis, that accounts for presence of confounding factors, as the most robust predictive biomarker of patient outcome in COVID-19.

Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment.

Extracellular HMGB1 triggers inflammation following infection or injury and supports tumorigenesis in inflammation-related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide-containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, diflunisal does not inhibit TLR4-dependent responses. Our findings clarify the mode of action of diflunisal and open the way to the rational design of functionally specific anti-inflammatory drugs.

High-mobility group box 1 protein orchestrates responses to tissue damage via inflammation, innate and adaptive immunity, and tissue repair.

A single protein, HMGB1, directs the triggering of inflammation, innate and adaptive immune responses, and tissue healing after damage. HMGB1 is the best characterized damage-associated molecular pattern (DAMP), proteins that are normally inside the cell but are released after cell death, and allow the immune system to distinguish between antigens that are dangerous or not. Notably, cells undergoing severe stress actively secrete HMGB1 via a dedicated secretion pathway: HMGB1 is relocated from the nucleus to the cytoplasm and then to secretory lysosomes or directly to the extracellular space. Extracellular HMGB1 (either released or secreted) triggers inflammation and adaptive immunological responses by switching among multiple oxidation states, which direct the mutually exclusive choices of different binding partners and receptors. Immune cells are first recruited to the damaged tissue and then activated; thereafter, HMGB1 supports tissue repair and healing, by coordinating the switch of macrophages to a tissue-healing phenotype, activation and proliferation of stem cells, and neoangiogenesis. Inevitably, HMGB1 also orchestrates the support of stressed but illegitimate tissues: tumors. Concomitantly, HMGB1 enhances the immunogenicity of mutated proteins in the tumor (neoantigens), promoting anti-tumor responses and immunological memory. Tweaking the activities of HMGB1 in inflammation, immune responses and tissue repair could bring large rewards in the therapy of multiple medical conditions, including cancer.

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma.

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

The Janus face of HMGB1 in heart disease: a necessary update.

High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein involved in transcription regulation, DNA replication and repair and nucleosome assembly. HMGB1 is passively released by necrotic tissues or actively secreted by stressed cells. Extracellular HMGB1 acts as a damage-associated molecular pattern (DAMPs) molecule and gives rise to several redox forms that by binding to different receptors and interactors promote a variety of cellular responses, including tissue inflammation or regeneration. Inhibition of extracellular HMGB1 in experimental models of myocardial ischemia/reperfusion injury, myocarditis, cardiomyopathies induced by mechanical stress, diabetes, bacterial infection or chemotherapeutic drugs reduces inflammation and is protective. In contrast, administration of HMGB1 after myocardial infarction induced by permanent coronary artery ligation ameliorates cardiac performance by promoting tissue regeneration. HMGB1 decreases contractility and induces hypertrophy and apoptosis in cardiomyocytes, stimulates cardiac fibroblast activities, and promotes cardiac stem cell proliferation and differentiation. Interestingly, maintenance of appropriate nuclear HMGB1 levels protects cardiomyocytes from apoptosis by preventing DNA oxidative stress, and mice with HMGB1cardiomyocyte-specific overexpression are partially protected from cardiac damage. Finally, higher levels of circulating HMGB1 are associated to human heart diseases. Hence, during cardiac injury, HMGB1 elicits both harmful and beneficial responses that may in part depend on the generation and stability of the diverse redox forms, whose specific functions in this context remain mostly unexplored. This review summarizes recent findings on HMGB1 biology and heart dysfunctions and discusses the therapeutic potential of modulating its expression, localization, and oxidative-dependent activities.

miR-34a Promotes Vascular Smooth Muscle Cell Calcification by Downregulating SIRT1 (Sirtuin 1) and Axl (AXL Receptor Tyrosine Kinase).

Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.

Loss of Endogenous HMGB2 Promotes Cardiac Dysfunction and Pressure Overload-Induced Heart Failure in Mice.

BACKGROUND: The rapid increase in the number of heart failure (HF) patients in parallel with the increase in the number of older people is receiving attention worldwide. HF not only increases mortality but decreases quality of life, creating medical and social problems. Thus, it is necessary to define molecular mechanisms underlying HF development and progression. HMGB2 is a member of the high-mobility group superfamily characterized as nuclear proteins that bind DNA to stabilize nucleosomes and promote transcription. A recent in vitro study revealed that HMGB2 loss in cardiomyocytes causes hypertrophy and increases HF-associated gene expression. However, it's in vivo function in the heart has not been assessed. Methods and Results: Western blotting analysis revealed increased HMGB2 expression in heart tissues undergoing pressure overload by transverse aorta constriction (TAC) in mice. Hmgb2 homozygous knockout (Hmgb2-/-) mice showed cardiac dysfunction due to AKT inactivation and decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a activity. Compared to wild-type mice, Hmgb2-/- mice had worsened cardiac dysfunction after TAC surgery, predisposing mice to HF development and progression. CONCLUSIONS: This study demonstrates that upregulation of cardiac HMGB2 is an adaptive response to cardiac stress, and that loss of this response could accelerate cardiac dysfunction, suggesting that HMGB2 plays a cardioprotective role.

Oxidative stress controls the choice of alternative last exons via a Brahma-BRCA1-CstF pathway.

Alternative splicing of terminal exons increases transcript and protein diversity. How physiological and pathological stimuli regulate the choice between alternative terminal exons is, however, largely unknown. Here, we show that Brahma (BRM), the ATPase subunit of the hSWI/SNF chromatin-remodeling complex interacts with BRCA1/BARD1, which ubiquitinates the 50 kDa subunit of the 3' end processing factor CstF. This results in the inhibition of transcript cleavage at the proximal poly(A) site and a shift towards inclusion of the distal terminal exon. Upon oxidative stress, BRM is depleted, cleavage inhibition is released, and inclusion of the proximal last exon is favoored. Our findings elucidate a novel regulatory mechanism, distinct from the modulation of transcription elongation by BRM that controls alternative splicing of internal exons.

Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance.

BACKGROUND: High Mobility Group Box 1 (HMGB1) was first identified as a nonhistone chromatin-binding protein that functions as a pro-inflammatory cytokine and a Damage-Associated Molecular Pattern molecule when released from necrotic cells or activated leukocytes. HMGB1 consists of two structurally similar HMG boxes that comprise the pro-inflammatory (B-box) and the anti-inflammatory (A-box) domains. Paradoxically, the A-box also contains the epitope for the well-characterized anti-HMGB1 monoclonal antibody "2G7", which also potently inhibits HMGB1-mediated inflammation in a wide variety of in vivo models. The molecular mechanisms through which the A-box domain inhibits the inflammatory activity of HMGB1 and 2G7 exerts anti-inflammatory activity after binding the A-box domain have been a mystery. Recently, we demonstrated that: 1) the TLR4/MD-2 receptor is required for HMGB1-mediated cytokine production and 2) the HMGB1-TLR4/MD-2 interaction is controlled by the redox state of HMGB1 isoforms. METHODS: We investigated the interactions of HMGB1 isoforms (redox state) or HMGB1 fragments (A- and B-box) with TLR4/MD-2 complex using Surface Plasmon Resonance (SPR) studies. RESULTS: Our results demonstrate that: 1) intact HMGB1 binds to TLR4 via the A-box domain with high affinity but an appreciable dissociation rate; 2) intact HMGB1 binds to MD-2 via the B-box domain with low affinity but a very slow dissociation rate; and 3) HMGB1 A-box domain alone binds to TLR4 more stably than the intact protein and thereby antagonizes HMGB1 by blocking HMGB1 from interacting with the TLR4/MD-2 complex. CONCLUSIONS: These findings not only suggest a model whereby HMGB1 interacts with TLR4/MD-2 in a two-stage process but also explain how the A-box domain and 2G7 inhibit HMGB1.

Correction to: Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance.

After publication of this article (He et al., 2018), the corresponding authors recognised an error in Scheme 1, in particular to section "A. HMGB1/TLR4/MD-2 complex formation". Above "Step 2: B box binding to MD-2", the text incorrectly read: "Low affinity / extremely slow off". In addition, some text was omitted below "TLR4/MD-2". The correct version of Scheme 1 is included in this Correction article. The original article (He et al., 2018) has been corrected.

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice.

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1-/- chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

The shedding-derived soluble receptor for advanced glycation endproducts sustains inflammation during acute Pseudomonas aeruginosa lung infection.

BACKGROUND: The membrane-bound isoform of the receptor for advanced glycation end products (FL-RAGE) is primarily expressed by alveolar epithelial cells and undergoes shedding by the protease ADAM10, giving rise to soluble cleaved RAGE (cRAGE). RAGE has been associated with the pathogenesis of several acute and chronic lung disorders. Whether the proteolysis of FL-RAGE is altered by a given inflammatory stimulus is unknown. Pseudomonas aeruginosa causes nosocomial infections in hospitalized patients and is the major pathogen associated with chronic lung diseases. METHODS: P. aeruginosa was injected in Rage-/- and wild-type mice and the impact on RAGE expression and shedding, levels of inflammation and bacterial growth was determined. RESULTS: Acute P. aeruginosa lung infection in mice induces a reduction of the active form of ADAM10, which determines an increase of FL-RAGE expression on alveolar cells and a concomitant decrease of pulmonary cRAGE levels. This was associated with massive recruitment of leukocytes and release of pro-inflammatory factors, tissue damage and relocation of cRAGE in the alveolar and bronchial cavities. The administration of sRAGE worsened bacterial burden and neutrophils infiltration. RAGE genetic deficiency reduced the susceptibility to P. aeruginosa infection, mitigating leukocyte recruitment, inflammatory molecules production, and bacterial growth. CONCLUSIONS: These data are the first to suggest that inhibition of FL-RAGE shedding, by affecting the FL-RAGE/cRAGE levels, is a novel mechanism for controlling inflammation to acute P. aeruginosa pneumonia. sRAGE in the alveolar space sustains inflammation in this setting. GENERAL SIGNIFICANCE: RAGE shedding may determine the progression of inflammatory lung diseases.

HMGB1 as biomarker and drug target.

High Mobility Group Box 1 protein was discovered as a nuclear protein, but it has a "second life" outside the cell where it acts as a damage-associated molecular pattern. HMGB1 is passively released or actively secreted in a number of diseases, including trauma, chronic inflammatory disorders, autoimmune diseases and cancer. Extracellular HMGB1 triggers and sustains the inflammatory response by inducing cytokine release and by recruiting leucocytes. These characteristics make extracellular HMGB1 a key molecular target in multiple diseases. A number of strategies have been used to prevent HMGB1 release or to inhibit its activities. Current pharmacological strategies include antibodies, peptides, decoy receptors and small molecules. Noteworthy, salicylic acid, a metabolite of aspirin, has been recently found to inhibit HMGB1. HMGB1 undergoes extensive post-translational modifications, in particular acetylation and oxidation, which modulate its functions. Notably, high levels of serum HMGB1, in particular of the hyper-acetylated and disulfide isoforms, are sensitive disease biomarkers and are associated with different disease stages. In the future, the development of isoform-specific HMGB1 inhibitors may potentiate and fine-tune the pharmacological control of inflammation. We review here the current therapeutic strategies targeting HMGB1, in particular the emerging and relatively unexplored small molecules-based approach.

Leukocyte HMGB1 is required for vessel remodeling in regenerating muscles.

Signals of tissue necrosis, damage-associated molecular patterns (DAMPs), cause inflammation. Leukocytes migrating into injured tissues tonically release DAMPs, including the high mobility group box 1 protein (HMGB1). In the absence of suitable models, the relative role of DAMPs released because of necrosis or leukocyte activation has not, so far, been dissected. We have generated a mouse model lacking Hmgb1 in the hematopoietic system and studied the response to acute sterile injury of the skeletal muscle. Regenerating fibers are significantly less numerous at earlier time points and smaller at the end of the process. Leukocyte Hmgb1 licenses the skeletal muscle to react to hypoxia, to express angiopoietin-2, and to initiate angiogenesis in response to injury. Vascularization of the regenerating tissue is selectively jeopardized in the absence of leukocyte Hmgb1, revealing that it controls the nutrient and oxygen supply to the regenerating tissue. Altogether, our results reveal a novel nonredundant role for leukocyte Hmgb1 in the repair of injured skeletal muscle.

5-Fluorouracil causes leukocytes attraction in the peritoneal cavity by activating autophagy and HMGB1 release in colon carcinoma cells.

Signals released by leukocytes contribute to tumor growth and influence the efficacy of antineoplastic treatments. The outcome of peritoneal carcinomatosis treatments is unsatisfactory, possibly because chemotherapy activates events that have in the long-term deleterious effects. In this study we offer evidence that 5-fluorouracile (5-FU), besides provoking apoptosis of MC38 colon carcinoma cells, induces a striking attraction of leukocytes both in an orthotopic model of colon carcinomatosis in vivo and in monocyte-migration assays in vitro. Leukocyte attraction depends on the presence of High Mobility Group Box 1 (HMGB1), an endogenous immune adjuvant and chemoattractant released by dying cells. Leukocyte recruitment is prevented in vivo and in vitro using blocking antibodies against HMGB1 and its competitive antagonist BoxA or by interfering with HMGB1 expression. Autophagy is required for leukocyte chemoattraction, since the latter abates upon pharmacological blockade of the autophagic flux while activation of autophagy per se, in the absence of death of colon carcinoma cells, is not sufficient to attract leukocytes. Our results identify autophagy induction and HMGB1 release in colon carcinoma cells as key events responsible for 5-FU elicited leukocyte attraction and define a novel rate-limiting target for combinatorial therapies.