TLR2, TLR4 and the MYD88 Signaling Pathway Are Crucial for Neutrophil Migration in Acute Kidney Injury Induced by Sepsis

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. the TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. the WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. the TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institute of Science and Technology (INCT)Universidade Federal de São Paulo, Dept Med, Disciplina Nefrol, São Paulo, BrazilUniv São Paulo, Dept Imunol, Lab Imunobiol Transplantes, São Paulo, BrazilHosp Israelita Albert Einstein, IIEP, São Paulo, BrazilUniv Fed Triangulo Mineiro, Uberaba, BrazilUniversidade Federal de São Paulo, Dept Med, Disciplina Nefrol, São Paulo, BrazilFAPESP: 07/07139-3Web of Scienc

INCREASED INFLAMMATION TRIGGERED BY HYPEROXALURIA EXACERBATES RENAL ISCHEMIA AND REPERCUSSION INJURY

Univ Fed Juiz De Fora, Div Nephrol, Juiz De Fora, BrazilHosp Israelita Albert Einstein, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista & Med, Lab Clin & Expt Immunol,Div Nephrol, Sao Paulo, BrazilUniv Sao Paulo, Dept Immunol, Lab Expt Immunobiol, Sao Paulo, BrazilUniv Fed Triangulo Mineiro, Uberaba, BrazilUniv Fed Sao Paulo, Escola Paulista & Med, Lab Clin & Expt Immunol,Div Nephrol, Sao Paulo, BrazilWeb of Scienc

Obesity increases blood-brain barrier permeability and aggravates the mouse model of multiple sclerosis

Obesity-induced insulin resistance (OIR) has been associated with an increased prevalence of neurodegenerative disorders such as multiple sclerosis. Obesity results in increased blood-brain barrier (BBB) permeability, specifically in the hypothalamic regions associated with the control of caloric intake. In obesity, the chronic state of low-grade inflammation has been implicated in several chronic autoimmune inflammatory disorders. However, the mechanisms that connect the inflammatory profile of obesity with the severity of experimental autoimmune encephalomyelitis (EAE) are poorly defined. In this study, we show that obese mice are more susceptible to EAE, presenting a worse clinical score with more severe pathological changes in the spinal cord when compared with control mice. Analysis of immune infiltrates at the peak of the disease shows that high-fat diet (HFD)- and control (chow)-fed groups do not present any difference in innate or adaptive immune cell compartments, indicating the increased severity occurs prior to disease onset. In the setting of worsening EAE in HFD-fed mice, we observed spinal cord lesions in myelinated regions and (blood brain barrier) BBB disruption. We also found higher levels of pro-inflammatory monocytes, macrophages, and IFN-γ+CD4+ T cells in the HFD-fed group compared to chow-fed animals. Altogether, our results indicate that OIR promotes BBB disruption, allowing the infiltration of monocytes/macrophages and activation of resident microglia, ultimately promoting CNS inflammation and exacerbation of EAE.</p

Expression of pro-inflammatory cytokines after CLP.

<p>mRNA expression of IL-1β (a), TNF-α (b), IL-6 (c), KC (d), IL-17 (e) and iNOS (f) in the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals per group. (g) Analysis of expression of α-IKK in kidney of WT, TLR2^−/− , TLR4^−/− and MyD88^−/− mice 24 hours after CLP. Results representative of a experiment with two animals/group Data shown as mean ± standard deviation (SD), * p<0.05, ** p<0.01 and *** p<0.001 vs WT.</p

Acute tubular necrosis in TLR2, TLR4 and MyD88 deficient mice subjected to CLP.

<p>Histology and quantification of ATN of control(a) and WT(b), TLR2^−/− (c), TLR4 ^−/− (d) and MyD88^−/− mice (e) 24 hours after CLP. Results of a representative experiment with 5 animals per group. Data shown as mean ± standard deviation (SD), *** p<0.0001 vs WT.</p

Endogenous ligands expression in kidney in animals subjected to CLP.

<p>mRNA expression of HMGB1 (a), HSP70 (b) in the kidney of WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals per group. Data presented as mean ± standard deviation (SD), * p<0.05 vs WT; ** p<0.01 vs WT.</p

Apoptosis in the kidney of TLR2, TLR4 and MyD88 deficient mice subjected to CLP.

<p>(<b>a</b>) Histological analysis of apoptosis by immunohistochemistry of cleaved caspase 3 of control (a), WT (b), TLR2 ^−/− (c), TLR4 ^−/− (d) and MyD88 ^−/− (e) mice respectively, 24 hours after CLP . Results of a representative experiment with 5 animals per group. (<b>b</b>) Histological analysis of apoptosis by immunofluorescence to TUNEL of control (a), WT (b), TLR2 ^−/− (c), TLR4 ^−/− (d) and MyD88 ^−/− mice (e) respectively, 24 hours after sepsis. (<b>c</b>) Score of cleaved caspase-3 in kidney represented in figure A. (<b>d</b>) Score of TUNEL in kidney represented in figure B. (<b>e</b>) Gene expression of BCL-2 in the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals/group. Data shown as mean ± standard deviation (SD), * p<0.05 vs WT and ** p<0.01 vs WT.</p

Neutrophil infiltration in renal tissue 24 hours after CLP.

<p>Gene expression of KC (a), IL-17 (b) , iNOS (c) in kidney 24 hours after CLP in WT, TLR2^−/− , TLR4^−/− and MyD88−/− mice. The mRNA was normalized to HPRT expression and compared to normal group. (d) Assay of myeloperoxidase activity in kidney of control, WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice 24 hours after CLP. (e) GR1 expression in the kidney of mice 24 hours after CLP. (f) Results of a representative experiment with five animals/group. Data shown as mean ± standard deviation (SD).* p<0.05 vs WT and ** p<0.01 vs WT. ND: not detected.</p

Migration GR1⁺ neutrophils into the peritoneal cavity after CLP.

<p>(a) Flow cytometry of the peritoneal cavity 24 hours after CLP in control, WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice. (b, c) Frequency of GR1^low and GR1^high cells population in control, WT, TLR2^−/−, TLR4^−/− and MyD88^−/− mice 24 hours after CLP. Data shown as mean ± standard deviation (SD).* p<0.05 vs. WT and ** p<0.01 vs. WT.</p