Transcriptomic responses from improved murine sepsis models can better mimic human surgical sepsis.

Historically, murine models of inflammation in biomedical research have been shown to minimally correlate with genomic expression patterns from blood leukocytes in humans. In 2019, our laboratory reported an improved surgical sepsis model of cecal ligation and puncture (CLP) that provides additional daily chronic stress (DCS), as well as adhering to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. This model phenotypically recapitulates the persistent inflammation, immunosuppression, and catabolism syndrome observed in adult human surgical sepsis survivors. Whether these phenotypic similarities between septic humans and mice are replicated at the circulating blood leukocyte transcriptome has not been demonstrated. Our analysis, in contrast with previous findings, demonstrated that genome-wide expression in our new murine model more closely approximated human surgical sepsis patients, particularly in the more chronic phases of sepsis. Importantly, our new model of murine surgical sepsis with chronic stress did not reflect well gene expression patterns from humans with community-acquired sepsis. Our work indicates that improved preclinical murine sepsis modeling can better replicate both the phenotypic and transcriptomic responses to surgical sepsis, but cannot be extrapolated to other sepsis etiologies. Importantly, these improved models can be a useful adjunct to human-focused and artificial intelligence-based forms of research in order to improve septic patients' morbidity and mortality.

Old Mice Demonstrate Organ Dysfunction as well as Prolonged Inflammation, Immunosuppression, and Weight Loss in a Modified Surgical Sepsis Model.

OBJECTIVES: Our goal was to "reverse translate" the human response to surgical sepsis into the mouse by modifying a widely adopted murine intra-abdominal sepsis model to engender a phenotype that conforms to current sepsis definitions and follows the most recent expert recommendations for animal preclinical sepsis research. Furthermore, we aimed to create a model that allows the study of aging on the long-term host response to sepsis. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Young (3-5 mo) and old (18-22 mo) C57BL/6j mice. INTERVENTIONS: Mice received no intervention or were subjected to polymicrobial sepsis with cecal ligation and puncture followed by fluid resuscitation, analgesia, and antibiotics. Subsets of mice received daily chronic stress after cecal ligation and puncture for 14 days. Additionally, modifications were made to ensure that "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" recommendations were followed. MEASUREMENTS AND MAIN RESULTS: Old mice exhibited increased mortality following both cecal ligation and puncture and cecal ligation and puncture + daily chronic stress when compared with young mice. Old mice developed marked hepatic and/or renal dysfunction, supported by elevations in plasma aspartate aminotransferase, blood urea nitrogen, and creatinine, 8 and 24 hours following cecal ligation and puncture. Similar to human sepsis, old mice demonstrated low-grade systemic inflammation 14 days after cecal ligation and puncture + daily chronic stress and evidence of immunosuppression, as determined by increased serum concentrations of multiple pro- and anti-inflammatory cytokines and chemokines when compared with young septic mice. In addition, old mice demonstrated expansion of myeloid-derived suppressor cell populations and sustained weight loss following cecal ligation and puncture + daily chronic stress, again similar to the human condition. CONCLUSIONS: The results indicate that this murine cecal ligation and puncture + daily chronic stress model of surgical sepsis in old mice adhered to current Minimum Quality Threshold in Pre-Clinical Sepsis Studies guidelines and met Sepsis-3 criteria. In addition, it effectively created a state of persistent inflammation, immunosuppression, and weight loss, thought to be a key aspect of chronic sepsis pathobiology and increasingly more prevalent after human sepsis.

Myeloid-derived suppressor cell function and epigenetic expression evolves over time after surgical sepsis.

BACKGROUND: Sepsis is an increasingly significant challenge throughout the world as one of the major causes of patient morbidity and mortality. Central to the host immunologic response to sepsis is the increase in circulating myeloid-derived suppressor cells (MDSCs), which have been demonstrated to be present and independently associated with poor long-term clinical outcomes. MDSCs are plastic cells and potentially modifiable, particularly through epigenetic interventions. The objective of this study was to determine how the suppressive phenotype of MDSCs evolves after sepsis in surgical ICU patients, as well as to identify epigenetic differences in MDSCs that may explain these changes. METHODS: Circulating MDSCs from 267 survivors of surgical sepsis were phenotyped at various intervals over 6 weeks, and highly enriched MDSCs from 23 of these samples were co-cultured with CD3/CD28-stimulated autologous T cells. microRNA expression from enriched MDSCs was also identified. RESULTS: We observed that MDSC numbers remain significantly elevated in hospitalized sepsis survivors for at least 6 weeks after their infection. However, only MDSCs obtained at and beyond 14 days post-sepsis significantly suppressed T lymphocyte proliferation and IL-2 production. These same MDSCs displayed unique epigenetic (miRNA) expression patterns compared to earlier time points. CONCLUSIONS: We conclude that in sepsis survivors, immature myeloid cell numbers are increased but the immune suppressive function specific to MDSCs develops over time, and this is associated with a specific epigenome. These findings may explain the chronic and persistent immune suppression seen in these subjects.

A Detailed Characterization of the Dysfunctional Immunity and Abnormal Myelopoiesis Induced by Severe Shock and Trauma in the Aged.

The elderly are particularly susceptible to trauma, and their outcomes are frequently dismal. Such patients often have complicated clinical courses and ultimately die of infection and sepsis. Recent research has revealed that although elderly subjects have increased baseline inflammation as compared with their younger counterparts, the elderly do not respond to severe infection or injury with an exaggerated inflammatory response. Initial retrospective analysis of clinical data from the Glue Grant trauma database demonstrated that despite a similar frequency, elderly trauma patients have worse outcomes to pneumonia than younger subjects do. Subsequent analysis with a murine trauma model also demonstrated that elderly mice had increased mortality after posttrauma Pseudomonas pneumonia. Blood, bone marrow, and bronchoalveolar lavage sample analyses from juvenile and 20-24-mo-old mice showed that increased mortality to trauma combined with secondary infection in the aged are not due to an exaggerated inflammatory response. Rather, they are due to a failure of bone marrow progenitors, blood neutrophils, and bronchoalveolar lavage cells to initiate and complete an emergency myelopoietic response, engendering myeloid cells that fail to clear secondary infection. In addition, elderly people appeared unable to resolve their inflammatory response to severe injury effectively.

Patterns of gene expression among murine models of hemorrhagic shock/trauma and sepsis.

Controversy remains whether the leukocyte genomic response to trauma or sepsis is dependent upon the initiating stimulus. Previous work illustrated poor correlations between historical models of murine trauma and sepsis (i.e., trauma-hemorrhage and lipopolysaccharide injection, respectively). The aim of this study is to examine the early genomic response in improved murine models of sepsis [cecal ligation and puncture (CLP)] and trauma [polytrauma (PT)] with and without pneumonia (PT+Pp). Groups of naïve, CLP, PT, and PT+Pp mice were killed at 2 h, 1 or 3 days. Total leukocytes were isolated for genome-wide expression analysis, and genes that were found to differ from control (false discovery rate adjusted P < 0.001) were assessed for fold-change differences. Spearman correlations were also performed. For all time points combined (CLP, PT, PT+Pp), there were 10,426 total genes that were found to significantly differ from naïve controls. At 2 h, the transcriptomic changes between CLP and PT showed a positive correlation (rs) of 0.446 (P < 0.0001) but were less positive thereafter. Correlations were significantly improved when we limited the analysis to common genes whose expression differed by a 1.5 fold-change. Both pathway and upstream analyses revealed the activation of genes known to be associated with pathogen-associated and damage-associated molecular pattern signaling, and early activation patterns of expression were very similar between polytrauma and sepsis at the earliest time points. This study demonstrates that the early leukocyte genomic response to sepsis and trauma are very similar in mice.

Protective immunity and defects in the neonatal and elderly immune response to sepsis.

Populations encompassing extremes of age, including neonates and elderly, have greater mortality from sepsis. We propose that the increased mortality observed in the neonatal and elderly populations after sepsis is due to fundamental differences in host-protective immunity and is manifested at the level of the leukocyte transcriptome. Neonatal (5-7 d), young adult (6-12 wk), or elderly (20-24 mo) mice underwent a cecal slurry model of intra-abdominal sepsis. Both neonatal and elderly mice exhibited significantly greater mortality to sepsis (p < 0.05). Neonates in particular exhibited significant attenuation of their inflammatory response (p < 0.05), as well as reductions in cell recruitment and reactive oxygen species production (both p < 0.05), all of which could be confirmed at the level of the leukocyte transcriptome. In contrast, elderly mice were also more susceptible to abdominal peritonitis, but this was associated with no significant differences in the magnitude of the inflammatory response, reduced bacterial killing (p < 0.05), reduced early myeloid cell activation (p < 0.05), and a persistent inflammatory response that failed to resolve. Interestingly, elderly mice expressed a persistent inflammatory and immunosuppressive response at the level of the leukocyte transcriptome, with failure to return to baseline by 3 d. This study reveals that neonatal and elderly mice have profoundly different responses to sepsis that are manifested at the level of their circulating leukocyte transcriptome, although the net result of increased mortality is similar. Considering these differences are fundamental aspects of the genomic response to sepsis, interventional therapies will require individualization based on the age of the population.

Aged mice are unable to mount an effective myeloid response to sepsis.

The elderly have increased morbidity and mortality following sepsis; however, the cause(s) remains unclear. We hypothesized that these poor outcomes are due in part to defects in innate immunity, rather than to an exaggerated early inflammatory response. Young (6-12 wk) or aged (20-24 mo) mice underwent polymicrobial sepsis, and subsequently, the aged mice had increased mortality and defective peritoneal bacterial clearance compared with young mice. No differences were found in the magnitude of the plasma cytokine responses. Although septic aged mice displayed equivalent or increased numbers of circulating, splenic, and bone marrow myeloid cells, some of these cells exhibited decreased phagocytosis, reactive oxygen species production, and chemotaxis. Blood leukocyte gene expression was less altered in aged versus young mice 1 d after sepsis. Aged mice had a relative inability to upregulate gene expression of pathways related to neutrophil-mediated protective immunity, chemokine/chemokine receptor binding, and responses to exogenous molecules. Expression of most MHC genes remained more downregulated in aged mice at day 3. Despite their increased myeloid response to sepsis, the increased susceptibility of aged mice to sepsis appears not to be due to an exaggerated inflammatory response, but rather, a failure to mount an effective innate immune response.

Improved emergency myelopoiesis and survival in neonatal sepsis by caspase-1/11 ablation.

Over one million newborns die annually from sepsis with the highest mortality in premature and low-birthweight infants. The inflammasome plays a central role in the regulation of innate immunity and inflammation, and is presumed to be involved in protective immunity, in large part through the caspase-1-dependent activation of interleukin-1ß (IL-1ß) and IL-18. Studies in endotoxic shock, however, suggest that endogenous caspase-1 activity and the inflammasome contribute to mortality primarily by promoting excessive systemic inflammatory responses. We examined whether caspase-1 and the inflammasome also regulate neonatal inflammation, host protective immunity and myelopoiesis during polymicrobial sepsis. Neonatal (5-7 days) C57BL/6 and caspase-1/11(-/-) mice underwent a low-lethality caecal slurry model of intra-abdominal sepsis (LD25-45 ). Ablation of caspase-1/11, but not apoptosis-associated speck-like protein containing a CARD domain or nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), improved neonatal survival following septic challenge compared with wild-type mice (P < 0·001), with decreased concentrations of inflammatory cytokines in the serum and peritoneum. Surprisingly, caspase-1/11(-/-) neonates also exhibited increased bone marrow and splenic haematopoietic stem cell expansion (P < 0·001), and increased concentrations of granulocyte and macrophage colony-stimulating factors in the peritoneum (P < 0·001) after sepsis. Ablation of caspase-1/11 signalling was also associated with increased recruitment of peritoneal macrophages and neutrophils (P < 0·001), increased phagocytosis by neutrophils (P = 0·003), and decreased bacterial colonization (P = 0·02) in the peritoneum. These findings suggest that endogenous caspase-1/11 activity, independent of the NLRP3 inflammasome, not only promotes the magnitude of the inflammatory response, but also suppresses protective immunity in the neonate, so contributing to innate immune dysfunction and poor survival in neonatal sepsis.

Maintenance of anti-Sm/RNP autoantibody production by plasma cells residing in ectopic lymphoid tissue and bone marrow memory B cells.

Although ectopic lymphoid tissue formation is associated with many autoimmune diseases, it is unclear whether it serves a functional role in autoimmune responses. 2,6,10,14-Tetramethylpentadecane causes chronic peritoneal inflammation and lupus-like disease with autoantibody production and ectopic lymphoid tissue (lipogranuloma) formation. A novel transplantation model was used to show that transplanted lipogranulomas retain their lymphoid structure over a prolonged period in the absence of chronic peritoneal inflammation. Recipients of transplanted lipogranulomas produced anti-U1A autoantibodies derived exclusively from the donor, despite nearly complete repopulation of the transplanted lipogranulomas by host lymphocytes. The presence of ectopic lymphoid tissue alone was insufficient, as an anti-U1A response was not generated by the host in the absence of ongoing peritoneal inflammation. Donor-derived anti-U1A autoantibodies were produced for up to 2 mo by plasma cells/plasmablasts recruited to the ectopic lymphoid tissue by CXCR4. Although CD4(+) T cells were not required for autoantibody production from the transplanted lipogranulomas, de novo generation of anti-U1A plasma cells/plasmablasts was reduced following T cell depletion. Significantly, a population of memory B cells was identified in the bone marrow and spleen that did not produce anti-U1A autoantibodies unless stimulated by LPS to undergo terminal differentiation. We conclude that 2,6,10,14-tetramethylpentadecane promotes the T cell-dependent development of class-switched, autoreactive memory B cells and plasma cells/plasmablasts. The latter home to ectopic lymphoid tissue and continue to produce autoantibodies after transplantation and in the absence of peritoneal inflammation. However, peritoneal inflammation appears necessary to generate autoreactive B cells de novo.

A better understanding of why murine models of trauma do not recapitulate the human syndrome.

OBJECTIVE: Genomic analyses from blood leukocytes have concluded that mouse injury poorly reflects human trauma at the leukocyte transcriptome. Concerns have focused on the modest severity of murine injury models, differences in murine compared with human age, dissimilar circulating leukocyte populations between species, and whether similar signaling pathways are involved. We sought to examine whether the transcriptomic response to severe trauma in mice could be explained by these extrinsic factors, by utilizing an increasing severity of murine trauma and shock in young and aged mice over time, and by examining the response in isolated neutrophil populations. DESIGN: Preclinical controlled in vivo laboratory study and retrospective cohort study. SETTING: Laboratory of Inflammation Biology and Surgical Science and multi-institution level 1 trauma centers. SUBJECTS: Six- to 10-week-old and 20- to 24-month-old C57BL/6 (B6) mice and two cohorts of 167 and 244 severely traumatized (Injury Severity Score > 15) adult (> 18 yr) patients. INTERVENTIONS: Mice underwent one of two severity polytrauma models of injury. Total blood leukocyte and neutrophil samples were collected. MEASUREMENTS AND MAIN RESULTS: Fold expression changes in leukocyte and neutrophil genome-wide expression analyses between healthy and injured mice (p < 0.001) were compared with human total and enriched blood leukocyte expression analyses of severe trauma patients at 0.5, 1, 4, 7, 14, and 28 days after injury (Glue Grant trauma-related database). We found that increasing the severity of the murine trauma model only modestly improved the correlation in the transcriptomic response with humans, whereas the age of the mice did not. In addition, the genome-wide response to blood neutrophils (rather than total WBC) was also not well correlated between humans and mice. However, the expression of many individual gene families was much more strongly correlated after injury in mice and humans. CONCLUSIONS: Although overall transcriptomic association remained weak even after adjusting for the severity of injury, age of the animals, timing, and individual leukocyte populations, there were individual signaling pathways and ontogenies that were strongly correlated between mice and humans. These genes are involved in early inflammation and innate/adaptive immunity.

Pleiotropic IFN-dependent and -independent effects of IRF5 on the pathogenesis of experimental lupus.

Genetic polymorphisms of IFN regulatory factor 5 (IRF5) are associated with an increased risk of lupus in humans. In this study, we examined the role of IRF5 in the pathogenesis of pristane-induced lupus in mice. The pathological response to pristane in IRF5(-/-) mice shared many features with type I IFN receptor (IFNAR)(-/-) and TLR7(-/-) mice: production of anti-Sm/RNP autoantibodies, glomerulonephritis, generation of Ly6C(hi) monocytes, and IFN-I production all were greatly attenuated. Lymphocyte activation following pristane injection was greatly diminished in IRF5(-/-) mice, and Th cell differentiation was deviated from Th1 in wild-type mice toward Th2 in IRF5(-/-) mice. Th cell development was skewed similarly in TLR7(-/-) or IFNAR(-/-) mice, suggesting that IRF5 alters T cell activation and differentiation by affecting cytokine production. Indeed, production of IFN-I, IL-12, and IL-23 in response to pristane was markedly decreased, whereas IL-4 increased. Unexpectedly, plasmacytoid dendritic cells (pDC) were not recruited to the site of inflammation in IRF5(-/-) or MyD88(-/-) mice, but were recruited normally in IFNAR(-/-) and TLR7(-/-) mice. In striking contrast to wild-type mice, pristane did not stimulate local expression of CCL19 and CCL21 in IRF5(-/-) mice, suggesting that IRF5 regulates chemokine-mediated pDC migration independently of its effects on IFN-I. Collectively, these data indicate that altered production of IFN-I and other cytokines in IRF5(-/-) mice prevents pristane from inducing lupus pathology by broadly affecting T and B lymphocyte activation/differentiation. Additionally, we uncovered a new, IFN-I-independent role of IRF5 in regulating chemokines involved in the homing of pDCs and certain lymphocyte subsets.

The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

Identification and description of a novel murine model for polytrauma and shock.

OBJECTIVE: To develop a novel polytrauma model that better recapitulates the immunologic response of the severely injured patient by combining long-bone fracture, muscle tissue damage, and cecectomy with hemorrhagic shock, resulting in an equivalent Injury Severity Score of greater than 15. We compared this new polytrauma/shock model to historically used murine trauma-hemorrhage models. DESIGN: Pre-clinical controlled in vivo laboratory study. SETTING: Laboratory of Inflammation Biology and Surgical Science. SUBJECTS: Six- to 10-week-old C57BL/6 (B6) mice. INTERVENTIONS: Mice underwent 90 minutes of shock (mean arterial pressure 30 mm Hg) and resuscitation via femoral artery cannulation followed by laparotomy (trauma-hemorrhage), hemorrhage with laparotomy and femur fracture, or laparotomy with cecetomy and femur fracture with muscle tissue damage (polytrauma). Mice were euthanized at 2 hours, 1 day, and 3 days postinjury. MEASUREMENTS AND MAIN RESULTS: The spleen, bone marrow, blood, and serum were collected from mice for analysis at the above time points. None of the models were lethal. Mice undergoing polytrauma exhibited a more robust inflammatory response with significant elevations in cytokine/chemokine concentrations when compared with traditional models. Polytrauma was the only model to induce neutrophilia (Ly6G (+)CD11b(+) cells) on days 1 and 3 (p<0.05). Polytrauma, as compared to trauma-hemorrhage and hemorrhage with laparotomy and femur fracture, induced a loss of circulating CD4(+) T cell with simultaneous increased cell activation (CD69(+) and CD25(+)), similar to human trauma. There was a prolonged loss of major histocompatibility complex class II expression on monocytes in the polytrauma model (p<0.05). Results were confirmed by genome-wide expression analysis that revealed a greater magnitude and duration of blood leukocyte gene expression changes in the polytrauma model than the trauma-hemorrhage and sham models. CONCLUSIONS: This novel polytrauma model better replicates the human leukocyte, cytokine, and overall inflammatory response following injury and hemorrhagic shock.

Sex differences associate with late microbiome alterations after murine surgical sepsis.

BACKGROUND: Sepsis-induced gut microbiome alterations contribute to sepsis-related morbidity and mortality. Given evidence for improved postsepsis outcomes in females compared with males, we hypothesized that female mice maintain microbiota resilience versus males. METHODS: Mixed-sex C57BL/6 mice underwent cecal ligation and puncture (CLP) with antibiotics, saline resuscitation, and daily chronic stress and were compared with naive (nonsepsis/no antibiotics) controls. For this work, the results of young (3-5 months) and old (18-22 months) adult mice were analyzed by sex, independent and dependent of age. Mice were sacrificed at days 7 and 14, and 16S rRNA gene sequencing was performed on fecal bacterial DNA. α and ß diversity were determined by Shannon index and Bray-Curtis with principal coordinate analysis, respectively. False discovery rate (FDR) correction was implemented to account for potential housing effect. RESULTS: In control mice, there was no difference in α or ß diversity between male and female mice (FDR, 0.76 and 0.99, respectively). However, male mice that underwent CLP with daily chronic stress had a decrease in microbiota α diversity at 7 days post-CLP (Shannon FDR, 0.005), which was sustained at 14 days post-CLP (Shannon FDR, 0.001), compared with baseline. In addition, male mice maintained differences in ß diversity even at day 14 compared with controls (FDR, <0.0001). In contrast, female mice had a decreased microbiota α diversity (Shannon FDR, 0.03) and ß diversity (FDR, 0.02) 7 days post-CLP but recovered their α and ß diversity by post-CLP day 14 (Shannon FDR, 0.5, and FDR, 0.02, respectively). Further analysis of females revealed that only young female mice were not different (ß diversity) post-CLP day 14 to controls. CONCLUSION: Although sepsis-induced perturbations of the intestinal microbiota occur initially in both male and female C57BL/6 mice, females demonstrate different microbiota by day 14. This may be seen primarily in younger females. This difference in recovery may play a role in outcome differences between sexes after sepsis.

Critical role for CXC ligand 10/CXC receptor 3 signaling in the murine neonatal response to sepsis.

Previous studies have suggested that neonates rely heavily on innate immunity for their antimicrobial response to bacterial infections. However, the innate immune response by neonates to bacterial infection remains poorly characterized. Here, we show that in a murine model of neonatal polymicrobial sepsis, CXC ligand 10 (CXCL10) concentrations increase in the blood and peritoneum concordant with the peritoneal recruitment of granulocytes and macrophages. Additionally, CXC receptor 3 (CXCR3) expression on elicited peritoneal macrophages and granulocytes increases following sepsis. Blockade of CXCL10 worsens not only recruitment and phagocytic function of peritoneal granulocytes and macrophages but also survival. Deletion of CXCR3 also significantly increases mortality to a septic challenge. Finally, we demonstrate that the protective adjuvant effect of pretreatment with a Toll-like receptor 4 agonist to neonatal sepsis is dependent on an endogenous CXCL10 response and that pretreatment of neonates with CXCL10 can also significantly improve macrophage and granulocyte function and modestly improve outcome to polymicrobial sepsis. Together, these data suggest a critical role for CXCL10 signaling during neonatal sepsis.

Pathogenic role of B cells in the development of diffuse alveolar hemorrhage induced by pristane.

Diffuse alveolar hemorrhage is an uncommon, yet often fatal, complication of systemic lupus erythematosus (SLE). Advances in the treatment of alveolar hemorrhage have been hampered because of the heterogeneity of clinical findings and the lack of suitable animal models. A single intraperitoneal injection of pristane induces a lupus-like syndrome characterized by lupus-related autoantibodies and glomerulonephritis in non-autoimmune-prone strains of mice. In addition, C57BL/6 (B6) mice frequently develop alveolar hemorrhage within a few weeks of pristane injection. Immunopathogenesis of pristane-induced alveolar hemorrhage was investigated in the present study. Early (2-4 weeks after injection) mortality due to hemorrhage was unique to C57BL/6 and C57BL/10 strains of mice. Recruitment of the macrophages and neutrophils preceded the hemorrhage by several days, and hemorrhage started 3-7 days after pristane injection in some mice, peaked at 2 weeks (84% in B6) and then resolved by 4 weeks in a majority of mice. Alveolar hemorrhage was independent of MyD88 (myeloid differentiation factor 88), or TLR7 pathways, in contrast to autoantibody production and glomerulonephritis, and was also independent of FcγR or Fas. Rag1(-/-) mice had a reduced prevalence of alveolar hemorrhage compared with B6 (P=0.01) congenics. However, T-cell receptor-deficient mice developed alveolar hemorrhage at a rate comparable to wild-type controls, whereas B6 Igµ(-/-) mice surprisingly had a strikingly reduced prevalence (7% vs 84% in B6, P<0.0001). Reconstitution of B6 Igµ(-/-) mice with wild-type B cells increased the prevalence to 50% (P=0.028). Pristane-induced alveolar hemorrhage is a useful model to study the pathogenesis and develop new therapy for this underappreciated and often life-threatening complication of SLE.

B cell proliferation, somatic hypermutation, class switch recombination, and autoantibody production in ectopic lymphoid tissue in murine lupus.

Intraperitoneal exposure of nonautoimmune mice to 2,6,10,14-tetramethylpentadecane (TMPD) causes lupus and the formation of ectopic lymphoid tissue. Although associated with humoral autoimmunity, it is not known whether Ab responses develop within ectopic lymphoid tissue or if B cells only secondarily migrate there. We show that ectopic lymphoid tissue induced by TMPD not only resembles secondary lymphoid tissue morphologically, but it also displays characteristics of germinal center reactions. Proliferating T and B lymphocytes were found within ectopic lymphoid tissue, activation-induced cytidine deaminase was expressed, and class-switched B cells were present. The presence of circular DNA intermediates, a hallmark of active class switch recombination, suggested that class switching occurs within the ectopic lymphoid tissue. Individual collections of ectopic lymphoid tissue ("lipogranulomas") from the same mouse contained different B cell repertoires, consistent with local germinal center-like reactions. Class-switched anti-RNP autoantibody-producing cells were also found in the lipogranulomas. Somatic hypermutation in the lipogranulomas was T cell-dependent, as was the production of isotype-switched anti-Sm/RNP autoantibodies. Thus, ectopic lymphoid tissue induced by TMPD recapitulates many of the functional characteristics of secondary lymphoid tissue and contains autoantibody-secreting cells, which may escape from normal censoring mechanisms in this location.

Aluminum Adjuvant Improves Survival Via NLRP3 Inflammasome and Myeloid Non-Granulocytic Cells in a Murine Model of Neonatal Sepsis.

ABSTRACT: Neonatal sepsis leads to significant morbidity and mortality with the highest risk of death occurring in preterm (<37 weeks) and low birth weight (<2,500 g) infants. The neonatal immune system is developmentally immature with well-described defects in innate and adaptive immune responses. Immune adjuvants used to enhance the vaccine response have emerged as potential therapeutic options, stimulating non-specific immunity and preventing sepsis mortality. Aluminum salts ("alum") have been used as immune adjuvants for over a century, but their mechanism of action remains poorly understood. This study aims to identify potential mechanisms by which pretreatment with alum induces host protective immunity to polymicrobial sepsis in neonatal mice. Utilizing genetic and cell-depletion studies, we demonstrate here that the prophylactic administration of aluminum adjuvants in neonatal mice improves sepsis survival via activation of the nucleotide oligomerization domain-like receptor family, pyrin-domain-containing 3 inflammasome and dendritic cell activation. Furthermore, this beneficial effect is dependent on myeloid, non-granulocytic Gr1-positive cells, and MyD88-signaling pathway activation. These findings suggest a promising therapeutic role for aluminum-based vaccine adjuvants to prevent development of neonatal sepsis and improve mortality in this highly vulnerable population.

Identification of unique microRNA expression patterns in bone marrow hematopoietic stem and progenitor cells after hemorrhagic shock and multiple injuries in young and old adult mice.

BACKGROUND: After severe trauma, the older host experiences more dysfunctional hematopoiesis of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs), and dysfunctional differentiation of circulating myeloid cells into effective innate immune cells. Our main objective was to compare BM HSPC microRNA (miR) responses of old and young mice in a clinically relevant model of severe trauma and shock. METHODS: C57BL/6 adult male mice aged 8 to 12 weeks (young) and 18 to 24 months (old) underwent multiple injuries and hemorrhagic shock (polytrauma [PT]) that engenders the equivalent of major trauma (Injury Severity Score, >15). Pseudomonas pneumonia (PNA) was induced in some young and old adult mice 24 hours after PT. MicroRNA expression patterns were determined from lineage-negative enriched BM HSPCs isolated from PT and PT-PNA mice at 24 and 48 hours postinjury, respectively. Genome-wide expression and pathway analyses were also performed on bronchoalveolar lavage (BAL) leukocytes from both mouse cohorts. RESULTS: MicroRNA expression significantly differed among all experimental conditions (p < 0.05), except for old-naive versus old-injured (PT or PT-PNA) mice, suggesting an inability of old mice to mount a robust early miR response to severe shock and injury. In addition, young adult mice had significantly more leukocytes obtained from their BAL, and there were greater numbers of polymorphonuclear cells compared with old mice (59.8% vs. 2.2%, p = 0.0069). Despite increased gene expression changes, BAL leukocytes from old mice demonstrated a more dysfunctional transcriptomic response to PT-PNA than young adult murine BAL leukocytes, as reflected in predicted upstream functional pathway analysis. CONCLUSION: The miR expression pattern in BM HSPCs after PT (+/-PNA) is dissimilar in old versus young adult mice. In the acute postinjury phase, old adult mice are unable to mount a robust miR HSPC response. Hematopoietic stem and progenitor cell miR expression in old PT mice reflects a diminished functional status and a blunted capacity for terminal differentiation of myeloid cells.

Septic Stability? Gut Microbiota in Young Adult Mice Maintains Overall Stability After Sepsis Compared to Old Adult Mice.

BACKGROUND: Older adults have worse outcomes after sepsis than young adults. Additionally, alterations of the gut microbiota have been demonstrated to contribute to sepsis-related mortality. We sought to determine if there were alterations in the gut microbiota with a novel sepsis model in old adult mice, which enter a state of persistent inflammation, immunosuppression, and catabolism (PICS), as compared with young adult mice, which recover with the sepsis model. METHODS: Mixed sex old (∼20 mo) and young (∼4 mo) C57Bl/6J mice underwent cecal ligation and puncture with daily chronic stress (CLP+DCS) and were compared with naive age-matched controls. Mice were sacrificed at CLP+DCS day 7 and feces collected for bacterial DNA isolation. The V3-V4 hypervariable region was amplified, 16S rRNA gene sequencing performed, and cohorts compared. α-Diversity was assessed using Chao1 and Shannon indices using rarefied counts, and ß-diversity was assessed using Bray-Curtis dissimilarity. RESULTS: Naive old adult mice had significantly different α and ß-diversity compared with naive adult young adult mice. After CLP+DCS, there was a significant shift in the α and ß-diversity (FDR = 0.03 for both) of old adult mice (naive vs. CLP+DCS). However, no significant shift was displayed in the microbiota of young mice that underwent CLP+DCS in regards to α-diversity (FDR = 0.052) and ß-diversity (FDR = 0.12), demonstrating a greater overall stability of their microbiota at 7 days despite the septic insult. The taxonomic changes in old mice undergoing CLP+DCS were dominated by decreased abundance of the order Clostridiales and genera Oscillospira. CONCLUSION: Young adult mice maintain an overall microbiome stability 7 days after CLP+DCS after compared with old adult mice. The lack of microbiome stability could contribute to PICS and worse long-term outcomes in older adult sepsis survivors. Further studies are warranted to elucidate mechanistic pathways and potential therapeutics.