Ana C. P. Souza

Class B Scavenger Receptor Types I and II and CD36 Mediate Bacterial Recognition and Proinflammatory Signaling Induced by Escherichia coli, Lipopolysaccharide, and Cytosolic Chaperonin 60

Class B scavenger receptors (SR-B) are lipoprotein receptors that also mediate pathogen recognition, phagocytosis, and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family, namely, CLA-1, CLA-2, and CD36, mediate recognition of bacteria not only through interaction with cell wall LPS but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3- to 5-fold) increased binding and endocytosis of Escherichia coli, LPS, and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS, and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36 > CLA-2 > CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II–null mice by 40–50 and 30–40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40–60% lower in CD36−/− relative to wild-type mice, whereas increased cytokine responses were found in SR-BI/II−/− mice. While investigating the discrepancy of in vitro versus in vivo data in SR-BI/II deficiency, SR-BI/II−/− mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in wild-type mice. SR-BI/II−/− mice with mineralocorticoid replacement demonstrated an ∼40–50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.

Erythropoietin prevents sepsis-related acute kidney injury in rats by inhibiting NF-κB and upregulating endothelial nitric oxide synthase

The pathophysiology of sepsis involves complex cytokine and inflammatory mediator networks, a mechanism to which NF-κB activation is central. Downregulation of endothelial nitric oxide synthase (eNOS) contributes to sepsis-induced endothelial dysfunction. Erythropoietin (EPO) has emerged as a major tissue-protective cytokine in the setting of stress. We investigated the role of EPO in sepsis-related acute kidney injury using a cecal ligation and puncture (CLP) model. Wistar rats were divided into three primary groups: control (sham-operated); CLP; and CLP+EPO. EPO (4,000 IU/kg body wt ip) was administered 24 and 1 h before CLP. Another group of rats received N-nitro-l-arginine methyl ester (l-NAME) simultaneously with EPO administration (CLP+EPO+l-NAME). A fifth group (CLP+EPOtreat) received EPO at 1 and 4 h after CLP. At 48 h postprocedure, CLP+EPO rats presented significantly higher inulin clearance than did CLP and CLP+EPO+l-NAME rats; hematocrit levels, mean arterial pressure, and metabolic balance remained unchanged in the CLP+EPO rats; and inulin clearance was significantly higher in CLP+EPOtreat rats than in CLP rats. At 48 h after CLP, creatinine clearance was significantly higher in the CLP+EPO rats than in the CLP rats. In renal tissue, pre-CLP EPO administration prevented the sepsis-induced increase in macrophage infiltration, as well as preserving eNOS expression, EPO receptor (EpoR) expression, IKK-α activation, NF-κB activation, and inflammatory cytokine levels, thereby increasing survival. We conclude that this protection, which appears to be dependent on EpoR activation and on eNOS expression, is attributable, in part, to inhibition of the inflammatory response via NF-κB downregulation.

Class B Scavenger Receptor Types I and II and CD36 Targeting Improves Sepsis Survival and Acute Outcomes in Mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II– or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50–70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II–deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II– and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.

Microparticles: markers and mediators of sepsis-induced microvascular dysfunction, immunosuppression, and AKI

Sepsis is a severe and complex syndrome that lacks effective prevention or therapeutics. The effects of sepsis on the microvasculature have become an attractive area for possible new targets and therapeutics. Microparticles (MPs) are cell membrane-derived particles that can promote coagulation, inflammation, and angiogenesis; and can participate in cell-to-cell communication. MPs retain cell membrane and cytoplasmic constituents of their parental cells, including two pro-coagulants: phosphatidylserine and tissue factor. We highlight the role of microparticles released by endothelial and circulating cells after sepsis-induced microvascular injury, and discuss possible mechanisms by which microparticles can contribute to endothelial dysfunction, immunosuppression, and multi-organ dysfunction--including sepsis-AKI. Once viewed as cellular byproducts, microparticles are emerging as a new class of markers and mediators in the pathogenesis of sepsis.

Automated quantification of renal fibrosis with Sirius Red and polarization contrast microscopy

Interstitial fibrosis is commonly measured by histology. The Masson trichrome stain is widely used, with semiquantitative scores subjectively assigned by trained operators. We have developed an objective technique combining Sirius Red staining, polarization contrast microscopy, and automated analysis. Repeated analysis of the same sections by the same operator (r = 0.99) or by different operators (r = 0.98) was highly consistent for Sirius Red, while Masson trichrome performed less consistently (r = 0.61 and 0.72, respectively). These techniques performed equally well when comparing sections from the left and right kidneys of mice. Poor correlation between Sirius Red and Masson trichrome may reflect different specificities, as enhanced birefringence with Sirius Red staining is specific for collagen type I and III fibrils. Combining whole‐section imaging and automated image analysis with Sirius Red/polarization contrast is a rapid, reproducible, and precise technique that is complementary to Masson trichrome. It also prevents biased selection of fields as fibrosis is measured on the entire kidney section. This new tool shall enhance our search for novel therapeutics and noninvasive biomarkers for fibrosis.

Comparison of serum creatinine and serum cystatin C as biomarkers to detect sepsis-induced acute kidney injury and to predict mortality in CD-1 mice

Acute kidney injury (AKI) dramatically increases sepsis mortality, but AKI diagnosis is delayed when based on serum creatinine (SCr) changes, due in part, to decreased creatinine production. During experimental sepsis, we compared serum cystatin C (sCysC), SCr, and blood urea nitrogen (BUN) to inulin glomerular filtration rate (iGFR) before or 3-18 h after cecal ligation and puncture (CLP)-induced sepsis in CD-1 mice. sCysC had a faster increase and reached peak levels more rapidly than SCr in both sepsis and bilateral nephrectomy (BiNx) models. sCysC was a better surrogate of iGFR than SCr during sepsis. Combining sCysC with SCr values into a composite biomarker improved correlation with iGFR better than any biomarker alone or any other combination. We determined the renal contribution to sCysC handling with BiNx. sCysC and SCr were lower post-BiNx/CLP than post-BiNx alone, despite increased inflammatory and nonrenal organ damage biomarkers. Sepsis decreased CysC production in nephrectomized mice without changing body weight or CysC space. Sepsis decreased sCysC production and increased nonrenal clearance, similar to effects of sepsis on SCr. sCysC, SCr, and BUN were measured 6 h postsepsis to link AKI with mortality. Mice with above-median sCysC, BUN, or SCr values 6 h postsepsis died earlier than mice with below-median values, corresponding to a substantial AKI association with sepsis mortality in this model. sCysC performs similarly to SCr in classifying mice at risk for early mortality. We conclude that sCysC detects AKI early and better reflects iGFR in CLP-induced sepsis. This study shows that renal biomarkers need to be evaluated in specific contexts.

TLR4 mutant mice are protected from renal fibrosis and chronic kidney disease progression

Chronic kidney disease (CKD) is associated with persistent low‐grade inflammation and immunosuppression. In this study we tested the role of Toll‐like receptor 4, the main receptor for endotoxin (LPS), in a mouse model of renal fibrosis and in a model of progressive CKD that better resembles the human disease. C3HeJ (TLR4 mutant) mice have a missense point mutation in the TLR4 gene, rendering the receptor nonfunctional. In a model of renal fibrosis after folic acid injection, TLR4 mutant mice developed less interstititial fibrosis in comparison to wild‐type (WT) mice. Furthermore, 4 weeks after 5/6 nephrectomy with continuous low‐dose angiotensin II infusion, C3HeOuJ (TLR4 WT) mice developed progressive CKD with albuminuria, increased serum levels of BUN and creatinine, glomerulosclerosis, and interstitial fibrosis, whereas TLR4 mutant mice were significantly protected from CKD progression. TLR4 WT mice also developed low‐grade systemic inflammation, splenocyte apoptosis and increased expression of the immune inhibitory receptor PD‐1 in the spleen, which were not observed in TLR4 mutant mice. In vitro, endotoxin (LPS) directly upregulated NLRP3 inflammasome expression in renal epithelial cells via TLR4. In summary, TLR4 contributes to renal fibrosis and CKD progression, at least in part, via inflammasome activation in renal epithelial cells, and may also participate in the dysregulated immune response that is associated with CKD.

Human SR-BI and SR-BII Potentiate Lipopolysaccharide-Induced Inflammation and Acute Liver and Kidney Injury in Mice

The class B scavenger receptors BI (SR-BI) and BII (SR-BII) are high-density lipoprotein receptors that recognize various pathogens, including bacteria and their products. It has been reported that SR-BI/II null mice are more sensitive than normal mice to endotoxin-induced inflammation and sepsis. Because the SR-BI/II knockout model demonstrates multiple immune and metabolic disorders, we investigated the role of each receptor in the LPS-induced inflammatory response and tissue damage using transgenic mice with pLiv-11–directed expression of human SR-BI (hSR-BI) or human SR-BII (hSR-BII). At 6 h after i.p. LPS injection, transgenic hSR-BI and hSR-BII mice demonstrated markedly higher serum levels of proinflammatory cytokines and 2- to 3-fold increased expression levels of inflammatory mediators in the liver and kidney, compared with wild-type (WT) mice. LPS-stimulated inducible NO synthase expression was 3- to 6-fold higher in the liver and kidney of both transgenic strains, although serum NO levels were similar in all mice. Despite the lower high-density lipoprotein plasma levels, both transgenic strains responded to LPS by a 5-fold increase of plasma corticosterone levels, which were only moderately lower than in WT animals. LPS treatment resulted in MAPK activation in tissues of all mice; however, the strongest response was detected for hepatic extracellular signal–regulated protein kinase 1 and 2 and kidney JNK of both transgenic mice. Histological examination of hepatic and renal tissue from LPS-challenged mice revealed more injury in hSR-BII, but not hSR-BI, transgenic mice versus WT controls. Our findings demonstrate that hSR-BII, and to a lesser extent hSR-BI, significantly increase LPS-induced inflammation and contribute to LPS-induced tissue injury in the liver and kidney, two major organs susceptible to LPS toxicity.

Human SR-BII mediates SAA uptake and contributes to SAA pro-inflammatory signaling in vitro and in vivo

Serum amyloid A (SAA) is an acute phase protein with cytokine-like and chemotactic properties, that is markedly up-regulated during various inflammatory conditions. Several receptors, including FPRL-1, TLR2, TLR4, RAGE, class B scavenger receptors, SR-BI and CD36, have been identified as SAA receptors. This study provides new evidence that SR-BII, splice variant of SR-BI, could function as an SAA receptor mediating its uptake and pro-inflammatory signaling. The uptake of Alexa Fluor488 SAA was markedly (~3 fold) increased in hSR-BII-expressing HeLa cells when compared with mock-transfected cells. The levels of SAA-induced interleukin-8 secretion by hSR-BII-expressing HEK293 cells were also significantly (~3–3.5 fold) higher than those detected in control cells. Moderately enhanced levels of phosphorylation of all three mitogen-activated protein kinases, ERK1/2, and p38 and JNK, were observed in hSR-BII-expressing cells following SAA stimulation when compared with control wild type cells. Transgenic mice with pLiv-11-directed liver/kidney overexpression of hSR-BI or hSR-BII were used to assess the in vivo role of each receptor in SAA-induced pro-inflammatory response in these organs. Six hours after intraperitoneal SAA injection both groups of transgenic mice demonstrated markedly higher (~2-5-fold) expression levels of inflammatory mediators in the liver and kidney compared to wild type mice. Histological examinations of hepatic and renal tissue from SAA-treated mice revealed moderate level of damage in the liver of both transgenic but not in the wild type mice. Activities of plasma transaminases, biomarkers of liver injury, were also moderately higher in hSR-B transgenic mice when compared to wild type mice. Our findings identify hSR-BII as a functional SAA receptor that mediates SAA uptake and contributes to its pro-inflammatory signaling via the MAPKs-mediated signaling pathways.

How can antibiotics worsen acute kidney injury but improve survival in experimental sepsis

Sepsis and septic shock are the most common causes of morbidity and mortality among critically ill patients, and remains the leading cause of acute kidney injury (AKI) among this population.1,2,3 Sepsis-induced AKI is independently associated with increased mortality and longer hospital stay.2, 4 AKI severity, as defined by RIFLE and AKIN criteria, parallels with increased hospital mortality in the general intensive care unit population.5 Despite much recent attention toward the interaction between sepsis and AKI, their relationship is still murky. Do patients die with AKI or of AKI? A seemingly straightforward study reported in this issue of Critical Care Medicine by Peng and colleagues now challenges the way we view sepsis-induced AKI in the ICUs, by showing that a commonly used antibiotic regimen temporarily worsens AKI severity while improving survival in experimental sepsis.6 This demonstrates a clear dissociation between AKI status and survival, which requires validation and re-examination in other settings in larger groups. The questions raised in this study could have substantial implications in the way that we view AKI endpoints, and follow-up studies are needed to examine the unintended effects of antibiotics and underlying mechanisms. Despite important new insights into the pathophysiology of sepsis, one of the cornerstones of sepsis treatment–the use of antibiotics together with volume resuscitation (and source control, if possible)–has not changed over the past four decades.7 Effective antibiotic administration within the first hour of documented hypotension is associated with increased survival to hospital discharge in adult patients with septic shock,8 and delays to appropriate antimicrobial therapy contribute to significant increases in the incidence of septic AKI.9 In a more contemporary cohort of patients with septic shock, AKI development was independently associated with delay to initiation of adequate antibiotics.10 The use of animal models to study sepsis requires balancing clinical relevance versus simplicity (easier to interpret) and practicality (easier to do). While use of antibiotics and fluid resuscitation are now standard practice in animal models, there is substantial variance between laboratories, probably due to differing adaptations of clinically relevant regimens. Peng et al. administered antibiotics 18 hours after cecal ligation and puncture (CLP) in rats, which is substantially later than the 6 hour time point that we and others have initiated antibiotics. As expected, Peng and collegues found that antibiotics improved survival, but paradoxically increased AKI at non-nephrotoxic doses. How could this happen? One avenue explored by Peng et al. is that within the context of sepsis, antibiotics increase AKI and inflammation along a liver damage and IL-6 axis. Do antibiotics improve survival because of AKI (and/or inflammation) or in spite of it? This dichotomy may depend on the dose, timing, and/or type of antibiotic administered. The authors raise the possibility that bactericidal antibiotics may release endotoxin (LPS) from bacteria, thus inducing inflammation.11 LPS and other bacterial constituents could activate Toll-like receptors (TLRs) and subsequent MyD88-dependent activation of the transcription factor NF-κB and production of cytokines. If inflammation is beneficial, then a proinflammatory cytokine such as TNFalpha12 could enhance the survival benefit from antibiotics. Conversely, an IL-6 based adjunctive anti-inflammatory therapy13 could improve survival if antibiotic-induced inflammation was an unavoidable side-effect of the bactericidal antibiotics. Of note, neither of these studies12,13 used antibiotics, and therefore the effects of these treatments could be different within the context of antibiotic treatment. Peng and colleagues point out an intriguing possibility from their data that antibiotic-treated CLP sepsis survivors recover from AKI and inflammation faster. Does the inflammation accelerate resolution of the infection? It probably depends on the context. Therefore, the clinical relevance of these findings will depend on the universality of the observations by Peng and colleagues. Do antibiotics increase AKI and inflammation while improving survival in other sepsis models such as pneumonia? Similarly, if these phenomena are observed in older animals with sepsis, or those with pre-existing co-morbidities such as chronic kidney disease14 these observations should have more direct translation to the aging patient population with sepsis. What if resolution of AKI15 is indeed more tightly associated with survival--than onset or peak of AKI is associated with mortality? The authors astutely point out that AKI biomarkers may not be adequate intermediate endpoints to develop therapeutic strategies for sepsis-AKI patients. In other words, AKI may not always be on the causal pathway to sepsis-induced mortality; therefore, we need biomarkers that distinguish lethal from ‘non-lethal/beneficial’ AKI.