Determination of the molecular and cellular mechanisms related to the immune response associated with the susceptibility of isogenic mice to sepsis

Sepse é definida como uma doença que acarreta disfunções de órgãos com risco a vida em decorrência de uma resposta inflamatória desregulada do hospedeiro à uma infecção. Estima-se uma ocorrência anual de 48,9 milhões de casos de sepse acarretando potencialmente 11 milhões de mortes em todo o mundo. Apesar de inúmeras tentativas de implementar imunoterapias que modulem moléculas relevantes na patogênese da sepse, até o momento nenhuma abordagem apresentou eficácia clínica. Assim, sugere-se que a patogênese da sepse não é completamente conhecida e mais estudos são necessários para a identificação de novos alvos terapêuticos. Neste sentido, nosso objetivo foi identificar os mecanismos moleculares e celulares da resposta imunológica que estão associados com a suscetibilidade de camundongos isogênicos, com mesma idade, sexo e mantidos nas mesmas condições ambientais, submetidos a sepse de mesma intensidade, e em decorrência identificar novos alvos para terapia. Inicialmente, observamos diferentes desfechos quando induzimos sepse de mesma intensidade pelo modelo de ligadura e perfuração do ceco (CLP) em camundongos isogênicos, do mesmo sexo, idade e criados nas mesmas condições ambientais. Embora não observarmos diferenças nas concentrações plasmáticas de citocinas após 6 horas da indução de sepse entre camundongos sobreviventes e não sobreviventes, nos tempos de 12 h, 24 h e 48 h, os camundongos que sobreviveram à sepse apresentaram concentrações reduzidas dessas citocinas, enquanto nos camundongos não sobreviventes essas concentrações permaneceram mais elevadas. De acordo com dados anteriormente publicados na literatura, observamos que camundongos não sobreviventes também apresentaram um aumento da concentração plasmática de biomarcadores de lesões hepáticas, renais e cardíacas e bacteremia. Além disso, estes camundongos apresentaram concentrações aumentadas de quimiocinas e citocinas nos pulmões, rins, coração, fígado e também no foco primário da infecção (cavidade peritoneal) 24h após CLP. Em seguida, isolamos leucócitos de camundongos sobreviventes e não sobrevivente após 12 h da indução de sepse e realizamos análise de single cell RNA sequencing. Através da avaliação dos dados obtidos por redução dimensional, identificamos várias populações de leucócitos e observarmos aumento da frequência de neutrófilos imaturos em camundongos não sobreviventes. Além disso, observamos que neutrófilos maduros de camundongos não sobreviventes apresentaram aumento da produção de mediadores próinflamatórios e expressão de Hif1a e Cd274, gene que codifica a proteína PD-L1. Na literatura é descrito que estas moléculas estão associadas com o aumento do tempo de sobrevivência de neutrófilos e aumento da produção de neutrophils extracellular traps, que provem lesões de órgãos. Além disso, é descrito que interferon gama (IFN-gama) é capaz de induzir a expressão de PD-L1 em neutrófilos e acarretar aumento da promoção de lesão pulmonar em modelo de endotoxemia por LPS. Neste sentido, demonstramos que INF-gama é capaz de induzir a expressão de Hif1a e PD-L1 em neutrófilos, e ainda que camundongos Ifng-deficientes são mais resistentes a sepse e apresentaram menor indução de PD-L1 em neutrófilos. Em conclusão, observamos que camundongos isogênicos que cresceram sob as mesmas condições ambientais, interessantemente apresentam diferentes suscetibilidades à sepse por CLP. Neste contexto, camundongos não sobreviventes apresentaram manutenção da elevada inflamação sistêmica e aumento de lesões de tecidos. Além disso, neutrófilos de camundongos não sobreviventes apresentam expressão aumentada de PD-L1 e Hif1a, que pode ser promovida pela sinalização de IFN-gama. Desta forma, a modulação negativa de IFN-gama, PD-L1 e Hif1a podem ser potenciais alvos terapêuticos para a sepse.Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. There is estimate an annual occurrence of 48.9 million cases of sepsis causing potentially 11 million deaths. Despite the attempt to implement immunotherapies modulating important molecules in the pathogenesis of sepsis, no approach showed efficacy in the clinics. This suggests that the pathogenesis of sepsis is not completely understood and that new methodologies may contribute to the identification of new therapeutic targets. In this context, the present work aimed to identify new therapeutic targets for sepsis analysis the molecular and cellular mechanisms related to the immune response responsible for the different outcomes of mice submitted to sepsis. Initially, by inducing the cecal ligation and puncture sepsis (CLP) in mice genetically equal and having the same grown-up environment, we observe different responses to sepsis in which half of the mice survive and the other half die from sepsis. Although we did not observe differences in the plasma concentrations of cytokines after 6 hours of sepsis induction between surviving and non-surviving mice, at 12 h, 24 h and 48 h, mice that survived sepsis showed reduced concentrations of these cytokines, while in non-survived mice these concentrations remained higher. Unsurprisingly, nonsurviving mice also showed increased plasma concentrations of biomarkers of liver, kidney and heart damage and bacteremia. Furthermore, these mice showed increased concentrations of chemokines and cytokines in the lungs, kidneys, heart, liver and also in the primary focus of infection (peritoneal cavity) 24h after CLP. Further, we isolated leukocytes from survived and non-survived mice after 12 h of sepsis induction and performed single cell RNA sequencing analysis. The evaluation of the data obtained by dimensional reduction revealed several populations of leukocytes, and we observed an increase in the frequency of immature neutrophils in non-survived mice. Furthermore, we observed that mature neutrophils from non-survived mice showed increased production of pro-inflammatory mediators and expression of Hif1a and Cd274, the gene that encodes the PD-L1 protein. In the literature it is described that these molecules are associated with increased neutrophil survival and increased production of neutrophil extracellular traps, which cause organ damage. Furthermore, it is described that interferon gamma (IFN-gamma) is able to induce PD-L1 expression in neutrophils and lead to increased promotion of lung injury in a model of endotoxemia by LPS. In this sense, we demonstrated that INF-gamma induced the expression of Hif1a and PD-L1 in neutrophils, and that Ifng-deficient mice are more resistant to sepsis and showed less induction of PD-L1 in neutrophils. In conclusion, we observed that isogenic mice grown under the same environmental conditions interestingly show different susceptibility to CLP sepsis. In this context, non-survived mice show maintenance of high systemic inflammation and increased tissue damage. Furthermore, neutrophils from non-survived mice show increased expression of PD-L1 and Hif1a, which can be promoted by IFN-gamma signaling. Thus, the negative modulation of IFN-gamma, PD-L1 and Hif1a may be potential therapeutic targets for sepsis

The role of inflammatory monocytes in sepsis

Sepse é uma síndrome, na qual, o paciente apresenta lesões de órgãos com risco a vida, em decorrência de uma inflamação exagerada desencadeada por uma infecção. Estima-se uma ocorrência anual de 31,5 milhões de casos de sepse e 19,4 milhões de casos de choque séptico no mundo, causando potencialmente 5,3 milhões de mortes. Esses índices alarmantes fizeram com que em 2017, a Organização Mundial da Saúde (OMS) adotasse uma resolução com o objetivo de aperfeiçoar a prevenção, diagnóstico e tratamento dessa condição clínica que vem sendo negligenciada. A iniciação da sepse, ocorre quando há um descontrole da infecção, acarretando excessiva ativação de células do sistema imune inato. Isso resulta em uma inflamação sistêmica danosa que é responsável pela maioria das alterações fisiopatológicas da sepse. Nesse contexto do sistema imune inato, o papel de neutrófilos já é bem compreendido da patogênese da sepse. Contudo, a função dos monócitos inflamatórios ainda não é bem estabelecida. Ao mesmo tempo que essas células podem participar do controle de infecções, elas também podem contribuir com a inflamação sistêmica e a lesão de órgãos. Deste modo, a compreensão do papel dessas células se faz importante para determinação de novos alvos terapêuticos para essa condição clínica. Nossos resultados demonstraram, em modelo experimental de sepse, que o aumento da emigração de monócitos inflamatórios da medula óssea está relacionado com maior taxa de mortalidade dos animais e exacerbação da inflamação sistêmica. A migração dessas células para órgãos, como rim e pulmão, está relacionado com inflamação e aumento de lesões, nesses locais. Deste modo, conclui-se que monócitos inflamatórios possuem um papel deletério na patogênese da sepseSepsis is a syndrome in which the patient has life-threatening organ damage due to an exaggerated inflammation triggered by an infection. The annual occurrence is 31.5 million cases of sepsis and 19.4 million cases of septic shock in the world, which potentially cause 5.3 million deaths. In concern of these alarming reports in 2017, the World Health Organization (WHO) adopted a resolution aimed at improving the prevention, diagnosis and treatment of this neglected clinical condition. The initiation of sepsis occurs when the infection was not controlled, causing excessive activation of the innate immune cells. This excessive activation causes a systemic inflammation that is responsible for most pathophysiological phenomena in sepsis. In this context of the innate immune system, the role of neutrophils is already well understood in the pathogenesis of sepsis. However, the role of inflammatory monocytes is not yet well established. These cells can participate in the control of infections, or can also contribute to systemic inflammation and organs damage. Thus, the understanding of the roles of these cells become important for the development of new therapeutic targets for this clinical condition. Our results demonstrated that the systemic increase of the inflammatory monocytes frequency is related to higher mortality rate, exacerbation of systemic inflammation, increased migration to organs (lung and kidney), and in these sites, are related to inflammation and lesions. Thus, we concluded that these cells have a deleterious role in the pathogenesis of sepsi

The role of inflammatory monocytes in sepsis

Sepse é uma síndrome, na qual, o paciente apresenta lesões de órgãos com risco a vida, em decorrência de uma inflamação exagerada desencadeada por uma infecção. Estima-se uma ocorrência anual de 31,5 milhões de casos de sepse e 19,4 milhões de casos de choque séptico no mundo, causando potencialmente 5,3 milhões de mortes. Esses índices alarmantes fizeram com que em 2017, a Organização Mundial da Saúde (OMS) adotasse uma resolução com o objetivo de aperfeiçoar a prevenção, diagnóstico e tratamento dessa condição clínica que vem sendo negligenciada. A iniciação da sepse, ocorre quando há um descontrole da infecção, acarretando excessiva ativação de células do sistema imune inato. Isso resulta em uma inflamação sistêmica danosa que é responsável pela maioria das alterações fisiopatológicas da sepse. Nesse contexto do sistema imune inato, o papel de neutrófilos já é bem compreendido da patogênese da sepse. Contudo, a função dos monócitos inflamatórios ainda não é bem estabelecida. Ao mesmo tempo que essas células podem participar do controle de infecções, elas também podem contribuir com a inflamação sistêmica e a lesão de órgãos. Deste modo, a compreensão do papel dessas células se faz importante para determinação de novos alvos terapêuticos para essa condição clínica. Nossos resultados demonstraram, em modelo experimental de sepse, que o aumento da emigração de monócitos inflamatórios da medula óssea está relacionado com maior taxa de mortalidade dos animais e exacerbação da inflamação sistêmica. A migração dessas células para órgãos, como rim e pulmão, está relacionado com inflamação e aumento de lesões, nesses locais. Deste modo, conclui-se que monócitos inflamatórios possuem um papel deletério na patogênese da sepseSepsis is a syndrome in which the patient has life-threatening organ damage due to an exaggerated inflammation triggered by an infection. The annual occurrence is 31.5 million cases of sepsis and 19.4 million cases of septic shock in the world, which potentially cause 5.3 million deaths. In concern of these alarming reports in 2017, the World Health Organization (WHO) adopted a resolution aimed at improving the prevention, diagnosis and treatment of this neglected clinical condition. The initiation of sepsis occurs when the infection was not controlled, causing excessive activation of the innate immune cells. This excessive activation causes a systemic inflammation that is responsible for most pathophysiological phenomena in sepsis. In this context of the innate immune system, the role of neutrophils is already well understood in the pathogenesis of sepsis. However, the role of inflammatory monocytes is not yet well established. These cells can participate in the control of infections, or can also contribute to systemic inflammation and organs damage. Thus, the understanding of the roles of these cells become important for the development of new therapeutic targets for this clinical condition. Our results demonstrated that the systemic increase of the inflammatory monocytes frequency is related to higher mortality rate, exacerbation of systemic inflammation, increased migration to organs (lung and kidney), and in these sites, are related to inflammation and lesions. Thus, we concluded that these cells have a deleterious role in the pathogenesis of sepsi

A Narrative Review of the Dorsal Root Ganglia and Spinal Cord Mechanisms of Action of Neuromodulation Therapies in Neuropathic Pain

Neuropathic pain arises from injuries to the nervous system in diseases such as diabetes, infections, toxicity, and traumas. The underlying mechanism of neuropathic pain involves peripheral and central pathological modifications. Peripheral mechanisms entail nerve damage, leading to neuronal hypersensitivity and ectopic action potentials. Central sensitization involves a neuropathological process with increased responsiveness of the nociceptive neurons in the central nervous system (CNS) to their normal or subthreshold input due to persistent stimuli, leading to sustained electrical discharge, synaptic plasticity, and aberrant processing in the CNS. Current treatments, both pharmacological and non-pharmacological, aim to alleviate symptoms but often face challenges due to the complexity of neuropathic pain. Neuromodulation is emerging as an important therapeutic approach for the treatment of neuropathic pain in patients unresponsive to common therapies, by promoting the normalization of neuronal and/or glial activity and by targeting cerebral cortical regions, spinal cord, dorsal root ganglia, and nerve endings. Having a better understanding of the efficacy, adverse events and applicability of neuromodulation through pre-clinical studies is of great importance. Unveiling the mechanisms and characteristics of neuromodulation to manage neuropathic pain is essential to understand how to use it. In the present article, we review the current understanding supporting dorsal root ganglia and spinal cord neuromodulation as a therapeutic approach for neuropathic pain

Haplotype Structures and Protein Levels of TGFB1 in HPV Infection and Cervical Lesion: A Case-Control Study

This study aimed to verify the role of TGFB1 variants (c.–1638G>A, c.–1347C>T, c.29C>T, and c.74G>C) in HPV infection susceptibility and cervical lesions development, and their impact on TGFB1 cervical and plasma levels. TGFB1 genotypes were assessed with PCR-RFLP and haplotypes were inferred for 190 HPV-uninfected and 161 HPV-infected women. TGFB1 levels were determined with immunofluorimetric assay. Case-control analyses were performed with logistic regression adjusted for possible confounders. Women carrying –1347TT or –1347CT+TT as well as those with 29CT, 29CC, or 29CT+CC were more likely to have HPV than –1347CC and 29TT carriers, respectively. Regarding haplotypes, the most frequent were *4 (GCTG) and *3 (GTCG). Women *4/*4 were less likely to have HPV than those with no *4 copy. Comparing the inheritance of *3 and *4, carriers of *3/*4 or *3/*3 were more susceptible to HPV than *4/*4. The TGFB1 plasma and cervical levels were higher in the infected patients. Plasma levels were also higher in infected women with low-grade lesions. HPV-infected patients carrying *3/Other and *3/Other+*3/*3 presented lower TGFB1 plasma levels than those with no copy of *3. TGFB1 variants could contribute to the comprehension of the TGFB1 role in HPV-caused cervical disease

CCR2-deficient mice are protected to sepsis by the disruption of the inflammatory monocytes emigration from the bone marrow

Abstract: Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Inflammatory monocytes are recruited to both the infection site and vital organs during sepsis; however, the mechanisms that orchestrate their migration, as well as the participation of these cells in systemic inflammation and vital organ damage, are still not fully elucidated. In this context, we described that CCR2-deficient mice had diminished migration of inflammatory monocytes from bone marrow to the circulation and subsequently to the site of infection and vital organs during cecal ligation and puncture (CLP)-induced polymicrobial sepsis. The reduction in the migration of inflammatory monocytes to the infection site was accompanied by a significant increase in the number of neutrophils in the same compartment, which seemed to counterbalance the absence of inflammatory monocytes in controlling microbial growth. Indeed, wild-type (WT) and CCR2-deficient mice under CLP presented similar control of infection. However, the CCR2-deficient mice were more resistant to sepsis, which was associated with a decrease in inflammatory mediators and organ damage biomarkers. Furthermore, the systemic adoptive transfer of CCR2-WT or CCR2-deficient inflammatory monocytes into CCR2-deficient mice equally increased the susceptibility to sepsis, demonstrating the deleterious role of these cells in the periphery even when CCR2 is absent. Thus, despite the host-protective role of inflammatory monocytes in controlling infection, our results demonstrated that the mechanism by which CCR2 deficiency shows protection to CLP-induced sepsis is due to a decrease of inflammatory monocytes emigration from bone marrow to the circulation and vital organs, resulting in the reduction of organ damage and systemic cytokine production

Resolvin D5 (RvD5) Reduces Renal Damage Caused by LPS Endotoxemia in Female Mice

In self-revolving gram-negative Escherichia coli infection, Resolvin D5 (RvD5) was found to enhance bacteria phagocytosis and reduce the production of inflammatory mediators, contributing to the resolution of infection. LPS (lipopolysaccharide) is a gram-negative bacterial structure product which activates the immune system and, at high doses, leads to endotoxemia. To our knowledge, the effect of RvD5 against LPS endotoxemia has not been investigated to date. Female Swiss mice received an i.p. treatment with RvD5 (0.1, 1 or 10 ng/animal). After 1 h, they were stimulated with LPS (10 mg/kg, i.v.), and samples were collected after additional 6 h. The resulting data demonstrated that RvD5 protected the kidneys (urea and creatinine serum levels) from tissue injury. These effects were related to an improvement in histopathological parameters and a reduction of enzymatic markers of leukocyte infiltration, pro-inflammatory cytokine (IL-1β, TNF-α, and IL-6) production, and oxidative stress. Antioxidant markers were also increased by RvD5, but IL-10 (an anti-inflammatory cytokine) levels were unaltered. We also observed that RvD5 reduced the infiltration of CD45+ hematopoietic cells into the kidneys, reduced the activation of NFκB and promoted the Nrf2 pathway by reducing Keap-1 levels. Our data indicate that RvD5 may be a therapeutic possibility to reduce kidney lesions in LPS endotoxemia