Izabela Galvão

A Role for Gut Microbiota and the Metabolite‐Sensing Receptor GPR43 in a Murine Model of Gout

Host–microbial interactions are central in health and disease. Monosodium urate monohydrate (MSU) crystals cause gout by activating the NLRP3 inflammasome, leading to interleukin‐1β (IL‐1β) production and neutrophil recruitment. This study was undertaken to investigate the relevance of gut microbiota, acetate, and the metabolite‐sensing receptor GPR43 in regulating inflammation in a murine model of gout.

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

Macrophage migration inhibitory factor drives neutrophil accumulation by facilitating IL-1β production in a murine model of acute gout.

This study evaluated the role of macrophage migration inhibitory factor in inflammation caused by monosodium urate crystals. The concentration of macrophage migration inhibitory factor was increased in synovial fluid of patients with acute gout, and there was a positive correlation between intra‐articular macrophage migration inhibitory factor and IL‐1β concentrations. In mice, the injection of monosodium urate crystals into the knee joint increased the levels of macrophage migration inhibitory factor in macrophages and in inflamed tissue. The injection of recombinant macrophage migration inhibitory factor into the joint of mice reproduced the inflammatory response observed in acute gout, including histologic changes, the recruitment of neutrophils, and increased levels of IL‐1β and CXCL1. Importantly, the accumulation of neutrophils and the amount IL‐1β in the joints were reduced in macrophage migration inhibitory factor‐deficient mice when injected with monosodium urate crystals. We observed a similar effect when we blocked macrophage migration inhibitory factor with (S,R)‐3‐(4‐hydroxyphenyl)‐4,5‐dihydro‐5‐isoxazole acetic acid or anti‐macrophage migration inhibitory factor. In addition, the blockade of IL‐1R and CXCR2 reduced recombinant macrophage migration inhibitory factor‐induced neutrophil recruitment. Mechanistically, recombinant macrophage migration inhibitory factor is important for the synthesis of il1β mRNA in vivo and in isolated macrophages. Altogether, macrophage migration inhibitory factor promotes neutrophil accumulation and is important for IL‐1β production, which are 2 crucial events contributing to the pathogenesis of acute gout.

Transmembrane TNF‐α is sufficient for articular inflammation and hypernociception in a mouse model of gout

Gout manifests as recurrent episodes of acute joint inflammation and pain due to the deposition of monosodium urate (MSU) crystals within the affected tissue in a process dependent on NLRP3 inflammasome activation. The synthesis, activation, and release of IL‐1β are crucial for MSU‐induced inflammation. The current study evaluated the mechanism by which TNF‐α contributed to MSU‐induced inflammation. Male C57BL/6J or transgenic mice were used in this study and inflammation was induced by the injection of MSU crystals into the joint. TNF‐α was markedly increased in the joint after the injection of MSU. There was inhibition in the infiltration of neutrophils, production of CXCL1 and IL‐1β, and decreased hypernociception in mice deficient for TNF‐α or its receptors. Pharmacological blockade of TNF‐α with Etanercept or pentoxyfylline produced similar results. Mechanistically, TNF‐α blockade resulted in lower amounts of IL‐1β protein and pro‐IL‐1β mRNA transcripts in joints. Gene‐modified mice that express only transmembrane TNF‐α had an inflammatory response similar to that of WT mice and blockade of soluble TNF‐α (XPro™1595) did not decrease MSU‐induced inflammation. In conclusion, TNF‐α drives expression of pro‐IL‐1β mRNA and IL‐1β protein in experimental gout and that its transmembrane form is sufficient to trigger MSU‐induced inflammation in mice.

Oral treatment with Bifidobacterium longum 51A reduced inflammation in a murine experimental model of gout

Gout is an acute inflammatory disease characterised by the presence of uric acid crystals in the joint. This event promotes neutrophil infiltration and activation that leads to tissue damage. We investigated here whether the oral administration of the probiotic strain Bifidobacterium longum 5(1A) (BL) could ameliorate monosodium urate crystal (MSU)-induced inflammation in a murine model of gout. Mice received oral administration of BL or saline daily for 7 days and then were injected with MSU in the knee cavity. Treatment with BL significantly alleviated the inflammatory parameters, as seen by reduced hypernociception, reduced neutrophil accumulation in the joint and myeloperoxidase activity in periarticular tissue. There was inhibition of the production of CXCL1 and interleukin(IL)-1β in joints. Levels of the anti-inflammatory cytokine IL-10 were significantly higher in the knee tissue of mice treated with than control mice injected with MSU. In conclusion, oral BL treatment reduced the inflammatory response in an experimental murine model of gout, suggesting it may be useful as an adjuvant treatment in patients with gout.

Annexin A1 promotes timely resolution of inflammation in murine gout

Gout is a self‐limited inflammatory disease caused by deposition of monosodium urate (MSU) crystals in the joints. Resolution of inflammation is an active process leading to restoration of tissue homeostasis. Here, we studied the role of Annexin A1 (AnxA1), a glucocorticoid‐regulated protein that has anti‐inflammatory and proresolving actions, in resolution of acute gouty inflammation. Injection of MSU crystals in the knee joint of mice induced inflammation that was associated with expression of AnxA1 during the resolving phase of inflammation. Neutralization of AnxA1 with antiserum or blockade of its receptor with BOC‐1 (nonselective) or WRW4 (selective) prevented the spontaneous resolution of gout. There was greater neutrophil infiltration after challenge with MSU crystals in AnxA1 knockout mice (AnxA1−/−) and delayed resolution associated to decreased neutrophil apoptosis and efferocytosis. Pretreatment of mice with AnxA1‐active N‐terminal peptide (Ac2–26) decreased neutrophil influx, IL‐1β, and CXCL1 production in periarticular joint. Posttreatment with Ac2–26 decreased neutrophil accumulation, IL‐1β, and hypernociception, and improved the articular histopathological score. Importantly, the therapeutic effects of Ac2–26 were associated with increased neutrophils apoptosis and shortened resolution intervals. In conclusion, AnxA1 plays a crucial role in the context of acute gouty inflammation by promoting timely resolution of inflammation.

Inhibition of Phosphodiesterase-4 during Pneumococcal Pneumonia Reduces Inflammation and Lung Injury in Mice

Pneumococcal pneumonia is a leading cause of mortality worldwide. The inflammatory response to bacteria is necessary to control infection, but it may also contribute to tissue damage. Phosphodiesterase-4 inhibitors, such as rolipram (ROL), effectively reduce inflammation. Here, we examined the impact of ROL in a pneumococcal pneumonia murine model. Mice were infected intranasally with 10(5)-10(6) CFU of Streptococcus pneumoniae, treated with ROL in a prophylactic or therapeutic schedule in combination, or not, with the antibiotic ceftriaxone. Inflammation and bacteria counts were assessed, and ex vivo phagocytosis assays were performed. ROL treatment during S. pneumoniae infection decreased neutrophil recruitment into lungs and airways and reduced lung injury. Prophylactic ROL treatment also decreased cytokine levels in the airways. Although modulation of inflammation by ROL ameliorated pneumonia, bacteria burden was not reduced. On the other hand, antibiotic therapy reduced bacteria without reducing neutrophil infiltration, cytokine level, or lung injury. Combined ROL and ceftriaxone treatment decreased lethality rates and was more efficient in reducing inflammation, by increasing proresolving protein annexin A1 (AnxA1) expression, and bacterial burden by enhancing phagocytosis. Lack of AnxA1 increased inflammation and lethality induced by pneumococcal infection. These data show that immunomodulatory effects of phosphodiesterase-4 inhibitors are useful during severe pneumococcal pneumonia and suggest their potential benefit as adjunctive therapy during infectious diseases.

Evaluating the effects of refined carbohydrate and fat diets with acute ethanol consumption using a mouse model of alcoholic liver injury

Alcoholism is a multifactorial and complex disorder responsible for 5.9% of deaths worldwide. Excessive consumption of ethanol (Et-OH) induces alcoholic liver disease (ALD), a condition comprising a spectrum of clinical signs and morphological changes, ranging from fatty liver (steatosis) to more severe forms of chronic liver injury. Secondary cofactors, such as nutritional and hepatotoxic comorbid conditions, can also contribute to liver disease development. Here we investigated the effects in the progression of ALD following short-term exposure to diet high in refined carbohydrates (HC), a high-sugar and -butter (HSB) hypercaloric diet and acute Et-OH consumption. HSB diet increased the body weight (BW) and adiposity independently of acute Et-OH consumption. HC diet did not affect BW but increased the adiposity, while acute Et-OH alone did not affect BW and adiposity. All groups of mice developed steatosis except the control group. Exposure to acute Et-OH and HSB diet increased the number of neutrophils and macrophages, and apoptosis in the liver. This combination also increased the number of circulating neutrophils and reduced mononuclear cells in the blood. Thus, short-term exposure to HSB diet and acute Et-OH intake is linked to increased liver injury. These findings offer important clues to understand the hepatic injuries associated with short exposure to hypercaloric diets and acute Et-OH.

The resolution of acute Inflammation induced by cyclic AMP is dependent on annexin A1.

Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.