Luciana P. Tavares

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.

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.

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.

Complement C5 activation during influenza A infection in mice contributes to neutrophil recruitment and lung injury

Influenza virus A (IAV) causes annual epidemics and intermittent pandemics that affect millions of people worldwide. Potent inflammatory responses are commonly associated with severe cases of IAV infection. The complement system, an important mechanism of innate and humoral immune responses to infections, is activated during primary IAV infection and mediates, in association with natural IgM, viral neutralization by virion aggregation and coating of viral hemmagglutinin. Increased levels of the anaphylatoxin C5a were found in patients fatally infected with the most recent H1N1 pandemic virus. In this study, our aim was to evaluate whether targeting C5 activation alters inflammatory lung injury and viral load in a murine model of IAV infection. To address this question C57Bl/6j mice were infected intranasally with 104 PFU of the mouse adapted Influenza A virus A/WSN/33 (H1N1) or inoculated with PBS (Mock). We demonstrated that C5a is increased in bronchoalveolar lavage fluid (BALF) upon experimental IAV infection. To evaluate the role of C5, we used OmCI, a potent arthropod-derived inhibitor of C5 activation that binds to C5 and prevents release of C5a by complement. OmCI was given daily by intraperitoneal injection from the day of IAV infection until day 5. Treatment with OmCI only partially reduced C5a levels in BALF. However, there was significant inhibition of neutrophil and macrophage infiltration in the airways, Neutrophil Extracellular Traps (NETs) formation, death of leukocytes, lung epithelial injury and overall lung damage induced by the infection. There was no effect on viral load. Taken together, these data suggest that targeting C5 activation with OmCI during IAV infection could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of IAV infections.

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δsse MGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and k cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δsse MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.

The Role and Effects of Glucocorticoid-Induced Leucine Zipper in the Context of Inflammation Resolution

Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate or mimic several actions of GC. This study assessed the role of GILZ in self-resolving and GC-induced resolution of neutrophilic inflammation induced by LPS in mice. GILZ expression was increased during the resolution phase of LPS-induced pleurisy, especially in macrophages with resolving phenotypes. Pretreating LPS-injected mice with trans-activator of transcription peptide (TAT)–GILZ, a cell-permeable GILZ fusion protein, shortened resolution intervals and improved resolution indices. Therapeutic administration of TAT-GILZ induced inflammation resolution, decreased cytokine levels, and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the activation of the survival-controlling proteins ERK1/2, NF-κB and Mcl-1. GILZ deficiency was associated with an early increase of annexin A1 (AnxA1) and did not modify the course of neutrophil influx induced by LPS. Dexamethasone treatment resolved inflammation and induced GILZ expression that was dependent on AnxA1. Dexamethasone-induced resolution was not altered in GILZ−/− mice due to compensatory expression and action of AnxA1. Our results show that therapeutic administration of GILZ efficiently induces a proapoptotic program that promotes resolution of neutrophilic inflammation induced by LPS. Alternatively, a lack of endogenous GILZ during the resolution of inflammation is compensated by AnxA1 overexpression.

Altered intracellular signaling cascades in peripheral blood mononuclear cells from BD patients

Bipolar disorder (BD) is a severe psychiatric disorder of complex physiopathology that has been associated with a pro-inflammatory state. The aim of the present study was to investigate intracellular pathways associated with inflammatory signaling, assessing the phosphorylation levels of transcription factor nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPKs) in peripheral blood mononuclear cells of euthymic BD patients and healthy controls. Fifteen BD euthymic type I patients, and 12 healthy controls matched by age and gender were enrolled in this study. All subjects were assessed by the Mini-International Neuropsychiatry Interview and the patients also by the Young Mania Rating Scale and the Hamilton Depression Rating Scale. Phosphorylation levels of p65 NF-κB subunit, and MAPK ERK1/2, and p38 were assessed by Western blot and flow cytometry. Plasma cytokines (IL-2, IL-4, IL6, IL-10, IFN-γ, TNF-α, and IL-17A) were measured using cytometric bead arrays. Western blot and flow cytometry analyses showed increased phosphorylation levels of p65 NF-κB subunit, and MAPKs ERK1/2, and p38 in BD patients in euthymia in comparison with controls. BD patients presented increased pro-inflammatory cytokines levels in comparison with controls, and TNF-α correlated with the levels of phosphorylated p65 NF-κB. The present study found increased activation of MAPK and NF-κB pathways in BD patients, which is in line with a pro-inflammatory status.

Control of Klebsiella pneumoniae pulmonary infection and immunomodulation by oral treatment with the commensal probiotic Bifidobacterium longum 5(1A).

Klebsiella pneumoniae (Kp) a common cause of pneumonia leads to intense lung injury and mortality that are correlated with infective exacerbations. Probiotics are a class of microorganisms that have immunomodulatory effects to benefit health. We investigated whether the probiotic Bifidobacterium longum 5(1A) induces protection in mice against lung infection induced by Kp and the potential involved mechanisms. Kp infection induced secretion of pro-inflammatory cytokines, neutrophil recruitment, significant bacterial load in the lung and 50% lethality. However, treatment with live B. longum 5(1A) induced faster resolution of inflammation associated with an increased production of IL-10, decreased lung damage with significantly reduction of bacterial burden that contributed to rescue 100% of mice from death. We found that these effects could be attributed, at least in part, to activation of the Toll-like receptor (TLR) adapter protein Mal, since B. longum 5(1A) treatment in Mal-deficient infected mice did not show the protection observed in wild type infected mice. Thus, we propose that live B. longum 5(1A) activates TLR-signaling pathway that results in ROS production and protects the host against pneumonia-induced death by finely tuning the inflammatory response and contributing to faster return to lung homeostasis.

Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals–induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.

Dietary fiber and the short-chain fatty acid acetate promote resolution of neutrophilic inflammation in a model of gout in mice

Gout is a disease characterized by the deposition of monosodium urate (MSU) crystals in the joints. Continuous gout episodes may lead to unresolved inflammatory responses and tissue damage. We investigated the effects of a high‐fiber diet and acetate, a short‐chain fatty acid (SCFA) resulting from the metabolism of fiber by gut microbiota, on the inflammatory response in an experimental model of gout in mice. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. The onset of inflammatory response induced by MSU crystals was not altered in animals given a high‐fiber diet, but the high‐fiber diet induced faster resolution of the inflammatory response. Similar results were obtained in animals given the SCFA acetate. Acetate was effective, even when given after injection of MSU crystals at the peak of the inflammatory response and induced caspase‐dependent apoptosis of neutrophils that accounted for the resolution of inflammation. Resolution of neutrophilic inflammation was associated with decreased NF‐κB activity and enhanced production of anti‐inflammatory mediators, including IL‐10, TGF‐β, and annexin A1. Acetate treatment or intake of a high‐fiber diet enhanced efferocytosis, an effect also observed in vitro with neutrophils treated with acetate. In conclusion, a high‐fiber diet or one of its metabolic products, acetate, controls the inflammatory response to MSU crystals by favoring the resolution of the inflammatory response. Our studies suggest that what we eat plays a determinant role in our capacity to fine tune the inflammatory response. Introduction