Remo Castro Russo

IL-33 Induces Antigen-Specific IL-5+ T Cells and Promotes Allergic-Induced Airway Inflammation Independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.

Regulation of leukocyte recruitment by the long pentraxin PTX3

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of 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)

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126–1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Annexin A1 modulates natural and glucocorticoid‐induced resolution of inflammation by enhancing neutrophil apoptosis

This study aimed at assessing whether AnxA1, a downstream mediator for the anti‐inflammatory effects of GCs, could affect the fate of immune cells in tissue exudates, using LPS‐induced pleurisy in BALB/c mice. AnxA1 protein expression in exudates was increased during natural resolution, as seen at 48–72 h post‐LPS, an effect augmented by treatment with GC and associated with marked presence of apoptotic neutrophils in the pleural exudates. The functional relevance of AnxA1 was determined using a neutralizing antibody or a nonspecific antagonist at FPR/ALXRs: either treatment inhibited both spontaneous and GC‐induced resolution of inflammation. Injection of Ac2‐26 (100 μg, given 4 h into the LPS response), an AnxA1‐active N‐terminal peptide, promoted active resolution and augmented the extent of neutrophil apoptosis. Such an effect was prevented by the pan‐caspase inhibitor zVAD‐fmk. Mechanistically, resolution of neutrophilic inflammation was linked to cell apoptosis with activation of Bax and caspase‐3 and inhibition of survival pathways Mcl‐1, ERK1/2, and NF‐κB. These novel in vivo data, using a dynamic model of acute inflammation, provide evidence that AnxA1 is a mediator of natural and GC‐induced resolution of inflammation with profound effects on neutrophil apoptosis.

Role of the Chemokine Receptor CXCR2 in Bleomycin-Induced Pulmonary Inflammation and Fibrosis

Pulmonary fibrosis is characterized by chronic inflammation and excessive collagen deposition. Neutrophils are thought to be involved in the pathogenesis of lung fibrosis. We hypothesized that CXCR2-mediated neutrophil recruitment is essential for the cascade of events leading to bleomycin-induced pulmonary fibrosis. CXCL1/KC was detected as early as 6 hours after bleomycin instillation and returned to basal levels after Day 8. Neutrophils were detected in bronchoalveolar lavage and interstitium from 12 hours and peaked at Day 8 after instillation. Treatment with the CXCR2 receptor antagonist, DF2162, reduced airway neutrophil transmigration but led to an increase of neutrophils in lung parenchyma. There was a significant reduction in IL-13, IL-10, CCL5/RANTES, and active transforming growth factor (TGF)-beta(1) levels, but not on IFN-gamma and total TGF-beta(1,) and enhanced granulocyte macrophage-colony-stimulating factor production in DF2162-treated animals. Notably, treatment with the CXCR2 antagonist led to an improvement of the lung pathology and reduced collagen deposition. Using a therapeutic schedule, DF2162 administered from Days 8 to 16 after bleomycin reduced pulmonary fibrosis and levels of active TGF-beta(1) and IL-13. DF2162 treatment reduced bleomycin-induced expression of von Willebrand Factor, a marker of angiogenesis, in the lung. In vitro, DF2162 reduced the angiogenic activity of IL-8 on human umbilical vein endothelial cells. In conclusion, we show that CXCR2 plays an important role in mediating fibrosis after bleomycin instillation. The compound blocks angiogenesis and the production of pro-angiogenic cytokines, and decreases IL-8-induced endothelial cell activation. An effect on neutrophils does not appear to account for the major effects of the blockade of CXCR2 in the system.

The CXCL8/IL-8 chemokine family and its receptors in inflammatory diseases.

Chemokines are small proteins that control several tissue functions, including cell recruitment and activation under homeostatic and inflammatory conditions. CXCL8 (interleukin-8) is a member of the chemokine family that acts on CXCR1 and CXCR2 receptors. CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL7 are also ELR+ chemokine members that bind to these receptors, especially CXCR2. The majority of studies on the biology of CXCL8 and their receptors have been performed in polymorphonuclear leukocytes. However, many other cells express CXCR1/CXCR2, including epithelial, endothelial, fibroblasts and neurons, contributing to the biological effects of CXCL8. There is substantial amount of experimental data suggesting that CXCL8 and receptors contribute to elimination of pathogens, but may also contribute significantly to disease-associated processes, including tissue injury, fibrosis, angiogenesis and tumorigenesis. Here, we discuss the biology of CXCL8 family and the potential therapeutic use of antagonists or blockers of these molecules in the context of organ-specific diseases.

PDE4 inhibition drives resolution of neutrophilic inflammation by inducing apoptosis in a PKA‐PI3K/Akt‐dependent and NF‐κB‐independent manner

PDE4 inhibitors are effective anti‐inflammatory drugs whose effects and putative mechanisms on resolution of inflammation and neutrophil apoptosis in vivo are still unclear. Here, we examined the effects of specific PDE4 inhibition on the resolution of neutrophilic inflammation in the pleural cavity of LPS‐challenged mice. LPS induced neutrophil recruitment that was increased at 4 h, peaked at 8–24 h, and declined thereafter. Such an event in the pleural cavity was preceded by increased levels of KC and MIP‐2 at 1 and 2 h. Treatment with the PDE4 inhibitor rolipram, at 4 h after LPS administration, decreased the number of neutrophils and increased the percentage of apoptotic cells in the pleural cavity in a PKA‐dependent manner. Conversely, delayed treatment with a CXCR2 antagonist failed to prevent neutrophil recruitment. Forskolin and db‐cAMP also decreased the number of neutrophils and increased apoptosis in the pleural cavity. The proapoptotic effect of rolipram was associated with decreased levels of the prosurvival protein Mcl‐1 and increased caspase‐3 cleavage. The pan‐caspase inhibitor zVAD‐fmk prevented rolipram‐induced resolution of inflammation. LPS resulted in a time‐dependent activation of Akt, which was blocked by treatment with rolipram or PI3K and Akt inhibitors, and PI3K and Akt inhibitors also enhanced apoptosis and promoted neutrophil clearance. Although LPS induced NF‐κB activation, which was blocked by rolipram, NF‐κB inhibitors did not promote resolution of neutrophil accumulation in this model. In conclusion, our data show that PDE4 inhibition resolves neutrophilic inflammation by promoting caspase‐dependent apoptosis of inflammatory cells by targeting a PKA/PI3K/Akt‐dependent survival pathway.

Tissue- and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo

PI3K plays a fundamental role in regulating neutrophil recruitment into sites of inflammation but the role of the different isoforms of PI3K remains unclear. In this study, we evaluated the role of PI3Kγ and PI3Kδ for neutrophil influx induced by the exogenous administration or the endogenous generation of the chemokine CXCL1. Administration of CXCL1 in PI3Kγ−/− or wild-type (WT) mice induced similar increases in leukocyte rolling, adhesion, and emigration in the cremaster muscle when examined by intravital microscopy. The induction of neutrophil recruitment into the pleural cavity or the tibia-femoral joint induced by the injection of CXCL1 was not significantly different in PI3Kγ−/− or WT mice. Neutrophil influx was not altered by treatment of WT mice with a specific PI3Kδ inhibitor, IC87114, or a specific PI3Kγ inhibitor, AS605240. The administration of IC87114 prevented CXCL1-induced neutrophil recruitment only in presence of the PI3Kγ inhibitor or in PI3Kγ−/− mice. Ag challenge of immunized mice induced CXCR2-dependent neutrophil recruitment that was inhibited by wortmannin or by blockade of and PI3Kδ in PI3Kγ−/− mice. Neutrophil recruitment to bronchoalveolar lavage induced by exogenously added or endogenous production of CXCL1 was prevented in PI3Kγ−/− mice. The accumulation of the neutrophils in lung tissues was significantly inhibited only in PI3Kγ−/− mice treated with IC87114. Neutrophil recruitment induced by exogenous administration of C5a or fMLP appeared to rely solely on PI3Kγ. Altogether, our data demonstrate that there is a tissue- and stimulus-dependent role of PI3Kγ and PI3Kδ for neutrophil recruitment induced by different chemoattractants in vivo.

Platelet-Activating Factor Receptor Plays a Role in Lung Injury and Death Caused by Influenza A in Mice

Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1+ cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans.