Gustavo B. Menezes

Intravascular Danger Signals Guide Neutrophils to Sites of Sterile Inflammation

Inflammation Response in Living Color Besides responding to microbial infection, our immune system also plays an important role in responding to sterile injury, for example, during trauma or organ necrosis. In a mouse model of sterile liver inflammation, McDonald et al. (p. 362) used dynamic in vivo imaging to visualize the innate immune response, which is dominated by neutrophils. Neutrophils were rapidly recruited to the site of inflammation through intravascular channels. Adenosine triphosphate generated from necrotic cells at the injury site and the Nlrp3 inflammasome were required for neutrophils to exit the circulation into the vascular endothelium, where they used integrins to adhere. A luminal chemokine gradient guided integrin-dependent, intravascular migration toward the site of injury. Finally, formyl peptides provided a signal to override the chemokine gradient and draw neutrophils into the site of injury. In vivo dynamic imaging reveals the underlying mechanisms of recruitment of neutrophils into injured tissue. Neutrophils are recruited from the blood to sites of sterile inflammation, where they contribute to wound healing but may also cause tissue damage. By using spinning disk confocal intravital microscopy, we examined the kinetics and molecular mechanisms of neutrophil recruitment to sites of focal hepatic necrosis in vivo. Adenosine triphosphate released from necrotic cells activated the Nlrp3 inflammasome to generate an inflammatory microenvironment that alerted circulating neutrophils to adhere within liver sinusoids. Subsequently, generation of an intravascular chemokine gradient directed neutrophil migration through healthy tissue toward foci of damage. Lastly, formyl-peptide signals released from necrotic cells guided neutrophils through nonperfused sinusoids into the injury. Thus, dynamic in vivo imaging revealed a multistep hierarchy of directional cues that guide neutrophil localization to sites of sterile inflammation.

Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure

Acetaminophen (APAP) is a safe analgesic and antipyretic drug. However, APAP overdose leads to massive hepatocyte death. Cell death during APAP toxicity occurs by oncotic necrosis, in which the release of intracellular contents can elicit a reactive inflammatory response. We have previously demonstrated that an intravascular gradient of chemokines and mitochondria‐derived formyl peptides collaborate to guide neutrophils to sites of liver necrosis by CXC chemokine receptor 2 (CXCR2) and formyl peptide receptor 1 (FPR1), respectively. Here, we investigated the role of CXCR2 chemokines and mitochondrial products during APAP‐induced liver injury and in liver neutrophil influx and hepatotoxicity. During APAP overdose, neutrophils accumulated into the liver, and blockage of neutrophil infiltration by anti–granulocyte receptor 1 depletion or combined CXCR2‐FPR1 antagonism significantly prevented hepatotoxicity. In agreement with our in vivo data, isolated human neutrophils were cytotoxic to HepG2 cells when cocultured, and the mechanism of neutrophil killing was dependent on direct contact with HepG2 cells and the CXCR2‐FPR1–signaling pathway. Also, in mice and humans, serum levels of both mitochondrial DNA (mitDNA) and CXCR2 chemokines were higher during acute liver injury, suggesting that necrosis products may reach remote organs through the circulation, leading to a systemic inflammatory response. Accordingly, APAP‐treated mice exhibited marked systemic inflammation and lung injury, which was prevented by CXCR2‐FPR1 blockage and Toll‐like receptor 9 (TLR9) absence (TLR9−/− mice). Conclusion: Chemokines and mitochondrial products (e.g., formyl peptides and mitDNA) collaborate in neutrophil‐mediated injury and systemic inflammation during acute liver failure. Hepatocyte death is amplified by liver neutrophil infiltration, and the release of necrotic products into the circulation may trigger a systemic inflammatory response and remote lung injury. (HEPATOLOGY 2012;56:1971–1982)

Hepatic DNA deposition drives drug‐induced liver injury and inflammation in mice

Drug‐induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self‐DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA‐rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll‐like receptor 9 (TLR9)‐mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up‐regulated TLR9 expression during acetaminophen‐mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF‐κB pathway. Likewise, adoptive transfer of wild‐type neutrophils to TLR9−/− mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. Conclusion: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue. (Hepatology 2015;61:348–360)

Selective Down-Regulation of Neutrophil Mac-1 in Endotoxemic Hepatic Microcirculation via IL-10

Hepatic neutrophil adhesion during endotoxemia is an integrin-independent, CD44-dependent process. Because integrins function in other endotoxemic vasculatures, we used spinning disk confocal intravital microscopy to assess whether LPS down-modulated integrin functions in sinusoids. First, we applied fMLP onto the liver surface, and compared it with systemic LPS administration. Local fMLP caused neutrophil adhesion, crawling, and emigration for at least 2 h. Surprisingly, the number of adherent and crawling neutrophils was markedly reduced in Mac-1−/− and ICAM-1−/− mice, but not in mice treated with anti-CD44 mAb. By contrast, systemic LPS injection induced a robust accumulation of neutrophils in sinusoids, which was dependent on CD44, but not on integrins. Strikingly, local fMLP could not induce any integrin-dependent adhesion in endotoxemic mice treated with anti-CD44 mAb, indicating that Mac-1-dependent neutrophil adhesion was inhibited by LPS. This response was localized to the hepatic microvasculature because neutrophils still adhered via integrins in brain microvasculature. ICAM-1/ICAM-2 levels were not decreased, but following LPS treatment, Mac-1 was down-regulated in neutrophils localized to liver, but not in the circulation. Mac-1 down-regulation in neutrophils was not observed in IL-10−/− mice. In vitro neutrophil incubation with IL-10 induced direct decrease of Mac-1 expression and adhesivity in LPS-stimulated neutrophils. Therefore, our data suggest that Mac-1 is necessary for neutrophil adhesion and crawling during local inflammatory stimuli in sinusoids, but during systemic inflammation, neutrophils are exposed to high concentrations of IL-10, leading to a CD44-dependent, integrin-independent adhesion. This may be a mechanism to keep neutrophils in sinusoids for intravascular trapping.

Different mechanisms underlie the analgesic actions of paracetamol and dipyrone in a rat model of inflammatory pain

The analgesics, paracetamol and dipyrone are weak inhibitors of the cyclooxygenase isoforms 1 or 2 (COX‐1, COX‐2) but more potent on COX‐3. Both are also weak anti‐inflammatory agents, relative to their analgesic and antipyretic activities. In a model of inflammatory pain mediated by prostaglandins, both compounds were analgesic. We have analysed this shared effect further in order to elucidate the underlying mechanisms.

Endothelial Expression of Guidance Cues in Vessel Wall Homeostasis Dysregulation Under Proatherosclerotic Conditions

Objective—Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues may regulate leukocyte trafficking into the vascular wall during atherogenesis. Approach and Results—We demonstrate that members of the netrin, semaphorin, and ephrin family of guidance molecules are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and semaphorin3A are expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes. Endothelial expression of these negative guidance cues is downregulated by proatherogenic factors, including oscillatory shear stress and proinflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show using intravital microscopy that inhibition of netrin-1 or semaphorin3A using blocking peptides increases leukocyte adhesion to the endothelium. Unlike netrin-1 and semaphorin3A, the guidance cue ephrinB2 is upregulated under proatherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines. Conclusions—The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking during atherogenesis.

Antiglaucomatous effects of the activation of intrinsic Angiotensin-converting enzyme 2.

PURPOSE To evaluate the effects of the activation of endogenous angiotensin-converting enzyme 2 (ACE2) using the compound diminazene aceturate (DIZE) in an experimental model of glaucoma in Wistar rats. METHODS DIZE (1 mg/kg) was administered daily, either systemically or topically, and the IOP was measured weekly. To examine the role of the Mas receptor in the effects of DIZE, the Ang-(1-7) antagonist A-779 was co-administered. Drainage of the aqueous humor was evaluated by using scintigraphy. The analysis of ACE2 expression by immunohistochemistry and the counting of retinal ganglion cells (RGCs) were performed in histologic sections. Additionally, the nerve fiber structure was evaluated by transmission electron microscopy. RESULTS The systemic administration and topical administration (in the form of eye drops) of DIZE increased the ACE2 expression in the eyes and significantly decreased the IOP of glaucomatous rats without changing the blood pressure. Importantly, this IOP-lowering action of DIZE was similar to the effects of dorzolamide. The antiglaucomatous effects of DIZE were blocked by A-779. Histologic analysis revealed that the reduction in the number of RGCs and the increase in the expression of caspase-3 in the RGC layer in glaucomatous animals were prevented by DIZE. This compound also prevented alterations in the cytoplasm of axons in glaucomatous rats. In addition to these neuroprotective effects, DIZE facilitated the drainage of the aqueous humor. CONCLUSIONS Our results evidence the pathophysiologic relevance of the ocular ACE2/Ang-(1-7)/Mas axis of the renin-angiotensin system and, importantly, indicate that the activation of intrinsic ACE2 is a potential therapeutic strategy to treat glaucoma.

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection.

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ−/− mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2−/− mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.

IL-33 targeting attenuates intestinal mucositis and enhances effective tumor chemotherapy in mice

Intestinal damage and severe diarrhea are serious side effects of cancer chemotherapy and constrain the usage of most such therapies. Here we show that interleukin-33 (IL-33) mediates the severe intestinal mucositis in mice treated with irinotecan (CPT-11), a commonly used cancer chemotherapeutic agent. Systemic CPT-11 administration led to severe mucosal damage, diarrhea, and body weight loss concomitant with the induction of IL-33 in the small intestine (SI). This mucositis was markedly reduced in mice deficient in the IL-33R (ST2−/−). Moreover, recombinant IL-33 exacerbated the CPT-11-induced mucositis, whereas IL-33 blockade with anti-IL-33 antibody or soluble ST2 markedly attenuated the disease. CPT-11 treatment increased neutrophil accumulation in the SI and adhesion to mesenteric veins. Supernatants from SI explants treated with CPT-11 enhanced transmigration of neutrophils in vitro in an IL-33-, CXCL1/2-, and CXCR2-dependent manner. Importantly, IL-33 blockade reduced mucositis and enabled prolonged CPT-11 treatment of ectopic CT26 colon carcinoma, leading to a beneficial outcome of the chemotherapy. These results suggest that inhibition of the IL-33/ST2 pathway may represent a novel approach to limit mucositis and thus improve the effectiveness of chemotherapy.

Dietary Supplementation with Omega-3-PUFA-Rich Fish Oil Reduces Signs of Food Allergy in Ovalbumin-Sensitized Mice

We investigated the effect of dietary supplementation with n-3 PUFA (fish oil source) in an experimental model of food allergy. Mice were sensitized (allergic group) or not (nonallergic group) with OVA and were fed with OVA diet to induce allergy signals. Mice were fed with regular diet in which 7% of lipid content was provided by soybean (5% of n-3 PUFA) or fish (25% of n-3 PUFA) oil. Allergic group mice had increased serum levels of antiovalbumin IgE and IgG1 and changes in small intestine, characterized by an increased edema, number of rolling leukocytes in microcirculation, eosinophil infiltration, mucus production, and Paneth cell degranulation, in comparison to non-allergic group. All these inflammatory parameters were reduced in mice fed high-n-3-PUFA diet. Our data together suggest that diet supplementation with n-3 PUFA from fish oil may consist of a valid adjuvant in food allergy treatment.