Loss of mTORC2 Activity in Neutrophils Impairs Fusion of Granules and Affects Cellular Metabolism Favoring Increased Bacterial Burden in Sepsis.

Sepsis is a complex infectious syndrome in which neutrophil participation is crucial for patient survival. Neutrophils quickly sense and eliminate the pathogen by using different effector mechanisms controlled by metabolic processes. The mammalian target of rapamycin (mTOR) pathway is an important route for metabolic regulation, and its role in neutrophil metabolism has not been fully understood yet, especially the importance of mTOR complex 2 (mTORC2) in the neutrophil effector functions. In this study, we observed that the loss of Rictor (mTORC2 scaffold protein) in primary mouse-derived neutrophils affects their chemotaxis by fMLF and their microbial killing capacity, but not the phagocytic capacity. We found that the microbicidal capacity was impaired in Rictor-deleted neutrophils because of an improper fusion of granules, reducing the hypochlorous acid production. The loss of Rictor also led to metabolic alterations in isolated neutrophils, increasing aerobic glycolysis. Finally, myeloid-Rictor-deleted mice (LysMRic Δ/Δ) also showed an impairment of the microbicidal capacity, increasing the bacterial burden in the Escherichia coli sepsis model. Overall, our results highlight the importance of proper mTORC2 activation for neutrophil effector functions and metabolism during sepsis.

A leukotriene-dependent spleen-liver axis drives TNF production in systemic inflammation.

Production of the proinflammatory cytokine tumor necrosis factor (TNF) must be precisely regulated for effective host immunity without the induction of collateral tissue damage. Here, we showed that TNF production was driven by a spleen-liver axis in a rat model of systemic inflammation induced by bacterial lipopolysaccharide (LPS). Analysis of cytokine expression and secretion in combination with splenectomy and hepatectomy revealed that the spleen generated not only TNF but also factors that enhanced TNF production by the liver, the latter of which accounted for nearly half of the TNF secreted into the circulation. Using mass spectrometry-based lipidomics, we identified leukotriene B4 (LTB4) as a candidate blood-borne messenger in this spleen-liver axis. LTB4 was essential for spleen-liver communication in vivo, as well as for humoral signaling between splenic macrophages and Kupffer cells in vitro. LPS stimulated the splenic macrophages to secrete LTB4, which primed Kupffer cells to secrete more TNF in response to LPS in a manner dependent on LTB4 receptors. These findings provide a framework to understand how systemic inflammation can be regulated at the level of interorgan communication.

Diet-induced obesity attenuates the hypothermic response to lipopolysaccharide independently of TNF-α production.

Life-threatening infections (sepsis) are usually associated with co-morbidities, among which obesity deserves attention. Here, we evaluated whether and how obesity affects the switch from fever to hypothermia that occurs in the most severe cases of sepsis, which is thought to provide physiological support for a change in host defense strategy from resistance to tolerance. Obesity was induced by keeping rats on a high-fat diet for 32-34 weeks. The hypothermia induced by a high dose of bacterial lipopolysaccharide (LPS, 300 µg/animal, i.a.) was attenuated in the obese rats, as compared to their low-fat diet counterparts. Surprisingly, such attenuation occurred in spite of an enhancement in the circulating level of TNF-α, the most renowned mediator of LPS-induced hypothermia. Hence, it seems that factors counteracting not the production, but rather the action of TNF-α are at play in rats with diet-induced obesity. One of these factors might be IL-1ß, a febrigenic mediator that also had its circulating levels augmented in the obese rats challenged with LPS. Taken together with previous reports of diet-induced obesity enhancing the fever induced by lower doses of LPS, the results of the present study indicate that obesity biases host defense toward a fever/resistance strategy, in lieu of a hypothermia/tolerance strategy.

Site-Specific Reprogramming of Macrophage Responsiveness to Bacterial Lipopolysaccharide in Obesity.

The mechanisms by which obesity may alter immune responses to pathogens are poorly understood. The present study assessed whether the intrinsic responsiveness of resident macrophages to bacterial lipopolysaccharide (LPS) is reprogrammed in high-fat diet (HFD)-induced obesity. Macrophages from adipose tissue, lung alveoli, and the peritoneal cavity were extracted from obese rats on a HFD or from their lean counterparts, and subsequently studied in culture under identical conditions. CD45+/CD68+ cells (macrophages) were abundant in all cultures, and became the main producers of TNF-α upon LPS stimulation. But although all macrophage subpopulations responded to LPS with an M1-like profile of cytokine secretion, the TNF-α/IL-10 ratio was the lowest in adipose tissue macrophages, the highest in alveolar macrophages, and intermediary in peritoneal macrophages. What is more, diet exerted qualitatively distinct effects on the cytokine responses to LPS, with obesity switching adipose tissue macrophages to a more pro-inflammatory program and peritoneal macrophages to a less pro-inflammatory program, while not affecting alveolar macrophages. Such reprogramming was not associated with changes in the inflammasome-dependent secretion of IL-1ß. The study further shows that the effects of diet on TNF-α/IL-10 ratios were linked to distinct patterns of NF-κB accumulation in the nucleus: while RelA was the NF-κB subunit most impacted by obesity in adipose tissue macrophages, cRel was the subunit affected in peritoneal macrophages. It is concluded that obesity causes dissimilar, site-specific changes in the responsiveness of resident macrophages to bacterial LPS. Such plasticity opens new avenues of investigation into the mechanisms linking obesity to pathogen-induced immune responses.

Aedes aegypti saliva impairs M1-associated proinflammatory phenotype without promoting or affecting M2 polarization of murine macrophages.

BACKGROUND: During the feeding process, the mouthparts of hematophagous mosquitoes break the skin barrier and probe the host tissue to find the blood. The saliva inoculated in this microenvironment modulates host hemostasis, inflammation and adaptive immune responses. However, the mechanisms involved in these biological activities remain poorly understood and few studies explored the potential roles of mosquito saliva on the individual cellular components of the immune system. Here, we report the immunomodulatory activities of Aedes aegypti salivary cocktail on murine peritoneal macrophages. RESULTS: The salivary gland extract (SGE) of Ae. aegypti inhibited the production of nitric oxide and inflammatory cytokines such as interleukin-6 (IL-6) and IL-12, as well as the expression of inducible nitric oxide synthase and NF-κB by murine macrophages stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). The spare respiratory capacity, the phagocytic and microbicidal activities of these macrophages were also reduced by Ae. aegypti SGE. These phenotypic changes are consistent with SGE suppressing the proinflammatory program of M1 macrophages. On the other hand, Ae. aegypti SGE did not influence M2-associated markers (urea production, arginase-1 and mannose receptor-1 expression), either in macrophages alternatively activated by IL-4 or in those classically activated by LPS plus IFN-γ. In addition, Ae. aegypti SGE did not display any cytokine-binding activity, nor did it affect macrophage viability, thus excluding supposed experimental artifacts. CONCLUSIONS: Given the importance of macrophages in a number of biological processes, our findings help to enlighten how vector saliva modulates vertebrate host immunity.

The inhibition of 5-Lipoxygenase (5-LO) products leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) modulates the inflammatory response and improves cutaneous wound healing.

To analyze the participation of the enzyme 5-lipoxygenase (5-LO) in skin repair, WT wounds were compared to those in 5-LO deficient mice (5-LO-/-), which presented faster closure and reduced inflammatory infiltrate in the skin, together with increased CD4 regulatory T cells markers in the draining lymph nodes. The 5-LO-/- wounds also had diminished TNF-α, CCL11, CCL7, CCL2, CXCL9, CCR1 and CXCR2 mRNA expression in the lesions, besides differential extracellular matrix remodeling. Furthermore, when cysteinyl leukotriene (cysLT) and leukotriene (LTB4) receptors were antagonized in WT mice, there was a remarkable reduction in TNF-α expression and faster skin healing, similarly to the findings in 5-LO-/- animals. Finally, our results suggested that 5-LO products, in special cysLT and LTB4, underline skin inflammation that follows skin injury and their neutralization may be an important strategy to improve cutaneous healing.

Elucidating the role of leptin in systemic inflammation: a study targeting physiological leptin levels in rats and their macrophages.

To elucidate the role of leptin in acute systemic inflammation, we investigated how its infusion at low, physiologically relevant doses affects the responses to bacterial lipopolysaccharide (LPS) in rats subjected to 24 h of food deprivation. Leptin was infused subcutaneously (0-20 µg·kg-1·h-1) or intracerebroventricularly (0-1 µg·kg-1·h-1). Using hypothermia and hypotension as biomarkers of systemic inflammation, we identified the phase extending from 90 to 240 min post-LPS as the most susceptible to modulation by leptin. In this phase, leptin suppressed the rise in plasma TNF-α and accelerated the recoveries from hypothermia and hypotension. Suppression of TNF-α was not accompanied by changes in other cytokines or prostaglandins. Leptin suppressed TNF-α when infused peripherally but not when infused into the brain. Importantly, the leptin dose that suppressed TNF-α corresponded to the lowest dose that limited food consumption; this dose elevated plasma leptin within the physiological range (to 5.9 ng/ml). We then conducted in vitro experiments to investigate whether an action of leptin on macrophages could parallel our in vivo observations. The results revealed that, when sensitized by food deprivation, LPS-stimulated peritoneal macrophages can be inhibited by leptin at concentrations that are lower than those reported to promote cytokine release. It is concluded that physiological levels of leptin do not exert a proinflammatory effect but rather an anti-inflammatory effect involving selective suppression of TNF-α via an action outside the brain. The mechanism of this effect might involve a previously unrecognized, suppressive action of leptin on macrophage subpopulations sensitized by food deprivation, but future studies are warranted.

Respiratory gas exchange as a new aid to monitor acidosis in endotoxemic rats: relationship to metabolic fuel substrates and thermometabolic responses.

This study introduces the respiratory exchange ratio (RER; the ratio of whole-body CO2 production to O2 consumption) as an aid to monitor metabolic acidosis during the early phase of endotoxic shock in unanesthetized, freely moving rats. Two serotypes of lipopolysaccharide (lipopolysaccharide [LPS] O55:B5 and O127:B8) were tested at shock-inducing doses (0.5-2 mg/kg). Phasic rises in RER were observed consistently across LPS serotypes and doses. The RER rise often exceeded the ceiling of the quotient for oxidative metabolism, and was mirrored by depletion of arterial bicarbonate and decreases in pH It occurred independently of ventilatory adjustments. These data indicate that the rise in RER results from a nonmetabolic CO2 load produced via an acid-induced equilibrium shift in the bicarbonate buffer. Having validated this new experimental aid, we asked whether acidosis was interconnected with the metabolic and thermal responses that accompany endotoxic shock in unanesthetized rats. Contrary to this hypothesis, however, acidosis persisted regardless of whether the ambient temperature favored or prevented downregulation of mitochondrial oxidation and regulated hypothermia. We then asked whether the substrate that fuels aerobic metabolism could be a relevant factor in LPS-induced acidosis. Food deprivation was employed to divert metabolism away from glucose oxidation and toward fatty acid oxidation. Interestingly, this intervention attenuated the RER response to LPS by 58%, without suppressing other key aspects of systemic inflammation. We conclude that acid production in unanesthetized rats with endotoxic shock results from a phasic activation of glycolysis, which occurs independently of physiological changes in mitochondrial oxidation and body temperature.

Spontaneous hypothermia in human sepsis is a transient, self-limiting, and nonterminal response.

Hypothermia in sepsis is generally perceived as something dysregulated and progressive although there has been no assessment on the natural course of this phenomenon in humans. This was the first study on the dynamics of hypothermia in septic patients not subjected to active rewarming, and the results were surprising. A sample of 50 subjects presenting with spontaneous hypothermia during sepsis was drawn from the 2005-2012 database of an academic hospital. Hypothermia was defined as body temperature below 36.0°C for longer than 2 h, with at least one reading of 35.5°C or less. The patients presented with 138 episodes of hypothermia, 21 at the time of the sepsis diagnosis and 117 with a later onset. However, hypothermia was uncommon in the final 12 h of life of the patients that succumbed. The majority (97.1%) of the hypothermic episodes were transient and self-limited; the median recovery time was 6 h; body temperature rarely fell below 34.0°C. Bidirectional oscillations in body temperature were evident in the course of hypothermia. Nearly half of the hypothermic episodes had onset in the absence of shock or respiratory distress, and the incidence of hypothermia was not increased during either of these conditions. Usage of antipyretic drugs, sedatives, neuroleptics, or other medications did not predict the onset of hypothermia. In conclusion, hypothermia appears to be a predominantly transient, self-limiting, and nonterminal phenomenon that is inherent to human sepsis. These characteristics resemble those of the regulated hypothermia shown to replace fever in animal models of severe systemic inflammation.

Factors associated to adherence to different treatment schemes with meglumine antimoniate in a clinical trial for cutaneous leishmaniasis.

The favorable outcome of the treatment of a disease is influenced by the adherence to therapy. Our objective was to assess factors associated with adherence to treatment of patients included in a clinical trial of equivalence between the standard and alternative treatment schemes with meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL), in the state of Rio de Janeiro. Between 2008 and 2011, 57 patients with CL were interviewed using a questionnaire to collect socioeconomic data. The following methods were used for adherence monitoring: counting of vial surplus, monitoring card, Morisky test and modified Morisky test (without the question regarding the schedule); we observed 82.1% (vial return), 86.0% (monitoring card), 66.7% (Morisky test) and 86.0% (modified Morisky test) adherence. There was a strong correlation between the method of vial counting and the monitoring card and modified Morisky test. A significant association was observed between greater adherence to treatment and low dose of MA, as well as with a lower number of people sleeping in the same room. We recommend the use of the modified Morisky test to assess adherence to treatment of CL with MA, because it is a simple method and with a good performance, when compared to other methods.

Factors associaeed to adherence to different tratment schemes with meglumine aantimoniate in a clinical trial for cutaneous leishmaniasis / Fatores associados à adesão a diferentes esquemas de tratamento com antimoniato de meglumina em ensaio clínico para leishmaniose cutânea

The favorable outcome of the treatment of a disease is influenced by the adherence to therapy. Our objective was to assess factors associated with adherence to treatment of patients included in a clinical trial of equivalence between the standard and alternative treatment schemes with meglumine antimoniate (MA) in the treatment of cutaneous leishmaniasis (CL), in the state of Rio de Janeiro. Between 2008 and 2011, 57 patients with CL were interviewed using a questionnaire to collect socioeconomic data. The following methods were used for adherence monitoring: counting of vial surplus, monitoring card, Morisky test and modified Morisky test (without the question regarding the schedule); we observed 82.1% (vial return), 86.0% (monitoring card), 66.7% (Morisky test) and 86.0% (modified Morisky test) adherence. There was a strong correlation between the method of vial counting and the monitoring card and modified Morisky test. A significant association was observed between greater adherence to treatment and low dose of MA, as well as with a lower number of people sleeping in the same room. We recommend the use of the modified Morisky test to assess adherence to treatment of CL with MA, because it is a simple method and with a good performance, when compared to other methods.

O desfecho favorável ao tratamento de uma enfermidade é influenciado pela adesão à terapia. Objetivamos avaliar fatores associados à adesão ao tratamento dos pacientes incluídos em ensaio clínico de equivalência entre o esquema de tratamento padrão e alternativos com antimoniato de meglumina (AM) no tratamento da leishmaniose cutânea (LC) no estado do Rio de Janeiro. Entre 2008 e 2011, 57 pacientes com LC foram entrevistados através de questionário para coleta de dados socioeconômicos. Para monitorização da adesão foram utilizados os seguintes métodos: contagem de ampolas excedentes, cartão de acompanhamento, teste de Morisky e teste de Morisky modificado (sem a pergunta referente ao horário). Observou-se adesão de 82,1% (devolução de ampolas), 86,0% (cartão de acompanhamento), 66,7% (teste de Morisky) e 86,0% (teste de Morisky modificado). Houve forte concordância entre o método contagem de ampolas e cartão de acompanhamento, bem como teste de Morisky modificado. Verificou-se associação significativa entre maior adesão ao tratamento e baixa dose de AM, bem como com menor número de pessoas dormindo no mesmo quarto. Recomendamos a utilização do teste de Morisky modificado na avaliação da adesão ao tratamento da LC com AM por ser método simples e com bom desempenho quando comparado aos outros testes.

Hypometabolism and hypothermia in the rat model of endotoxic shock: independence of circulatory hypoxia.

We tested the hypothesis that development of hypothermia instead of fever in endotoxic shock is consequential to hypoxia. Endotoxic shock was induced by bacterial lipopolysaccharide (LPS, 500 µg kg(-1) i.v.) in rats at an ambient temperature of 22 °C. A ß3-adrenergic agonist known to activate metabolic heat production, CL316,243, was employed to evaluate whether thermogenic capacity could be impaired by the fall in oxygen delivery (DO2) during endotoxic shock. This possibility was rejected as CL316,243 (0.15 mg kg(-1) i.v.) evoked similar rises in oxygen consumption (V̇O2) in the presence and absence of endotoxic shock. Next, to investigate whether a less severe form of circulatory hypoxia could be triggering hypothermia, the circulating volume of LPS-injected rats was expanded using 6% hetastarch with the intention of improving tissue perfusion and alleviating hypoxia. This intervention attenuated not only the fall in arterial pressure induced by LPS, but also the associated falls in V̇O2 and body temperature. These effects, however, occurred independently of hypoxia, as they were not accompanied by any detectable changes in NAD(+)/NADH ratios. Further experimentation revealed that even the earliest drops in cardiac output and DO2 during endotoxic shock did not precede the reduction in V̇O2 that brings about hypothermia. In fact, DO2 and V̇O2 fell in such a synchrony that the DO2/V̇O2 ratio remained unaffected. Only when hypothermia was prevented by exposure to a warm environment (30 °C) did an imbalance in the DO2/V̇O2 ratio become evident, and such an imbalance was associated with reductions in the renal and hypothalamic NAD(+)/NADH ratios. In conclusion, hypometabolism and hypothermia in endotoxic shock are not consequential to hypoxia but serve as a pre-emptive strategy to avoid hypoxia in this model.