IRF3 Signaling within the Mouse Stroma Influences Sepsis Pathogenesis.

IFN regulatory factor 3 (IRF3) is a transcription factor that is activated by multiple pattern-recognition receptors. We demonstrated previously that IRF3 plays a detrimental role in a severe mouse model of sepsis, induced by cecal ligation and puncture. In this study, we found that IRF3-knockout (KO) mice were greatly protected from sepsis in a clinically relevant version of the cecal ligation and puncture model incorporating crystalloid fluids and antibiotics, exhibiting improved survival, reduced disease score, lower levels of serum cytokines, and improved phagocytic function relative to wild-type (WT) mice. Computational modeling revealed that the overall complexity of the systemic inflammatory/immune network was similar in IRF3-KO versus WT septic mice, although the tempo of connectivity differed. Furthermore, the mediators driving the network differed: TNF-α, IL-1ß, and IL-6 predominated in WT mice, whereas MCP-1 and IL-6 predominated in IRF3-KO mice. Network analysis also suggested differential IL-6-related inflammatory programs in WT versus IRF3-KO mice. We created bone marrow chimeras to test the role of IRF3 within leukocytes versus stroma. Surprisingly, chimeras with IRF3-KO bone marrow showed little protection from sepsis, whereas chimeras with IRF3-KO stroma showed a substantial degree of protection. We found that WT and IRF3-KO macrophages had a similar capacity to produce IL-6 and phagocytose bacteria in vitro. Adoptive transfer experiments demonstrated that the genotype of the host environment affected the capacity of monocytes to produce IL-6 during sepsis. Thus, IRF3 acts principally within the stromal compartment to exacerbate sepsis pathogenesis via differential impacts on IL-6-related inflammatory programs.

mPR-Specific Actions Influence Maintenance of the Blood-Brain Barrier (BBB).

Cerebral cavernous malformations (CCMs) are characterized by abnormally dilated intracranial microvascular sinusoids that result in increased susceptibility to hemorrhagic stroke. It has been demonstrated that three CCM proteins (CCM1, CCM2, and CCM3) form the CCM signaling complex (CSC) to mediate angiogenic signaling. Disruption of the CSC will result in hemorrhagic CCMs, a consequence of compromised blood-brain barrier (BBB) integrity. Due to their characteristically incomplete penetrance, the majority of CCM mutation carriers (presumed CCM patients) are largely asymptomatic, but when symptoms occur, the disease has typically reached a clinical stage of focal hemorrhage with irreversible brain damage. We recently reported that the CSC couples both classic (nuclear; nPRs) and nonclassic (membrane; mPRs) progesterone (PRG)-receptors-mediated signaling within the CSC-mPRs-PRG (CmP) signaling network in nPR(-) breast cancer cells. In this report, we demonstrate that depletion of any of the three CCM genes or treatment with mPR-specific PRG actions (PRG/mifepristone) results in the disruption of the CmP signaling network, leading to increased permeability in the nPR(-) endothelial cells (ECs) monolayer in vitro. Finally, utilizing our in vivo hemizygous Ccm mutant mice models, we demonstrate that depletion of any of the three CCM genes, in combination with mPR-specific PRG actions, is also capable of leading to defective homeostasis of PRG in vivo and subsequent BBB disruption, allowing us to identify a specific panel of etiological blood biomarkers associated with BBB disruption. To our knowledge, this is the first report detailing the etiology to predict the occurrence of a disrupted BBB, an indication of early hemorrhagic events.

The circadian clock controls toll-like receptor 9-mediated innate and adaptive immunity.

Circadian rhythms refer to biologic processes that oscillate with a period of ~24 hr. These rhythms are sustained by a molecular clock and provide a temporal matrix that ensures the coordination of homeostatic processes with the periodicity of environmental challenges. We demonstrate the circadian molecular clock controls the expression and function of Toll-like receptor 9 (TLR9). In a vaccination model using TLR9 ligand as adjuvant, mice immunized at the time of enhanced TLR9 responsiveness presented weeks later with an improved adaptive immune response. In a TLR9-dependent mouse model of sepsis, we found that disease severity was dependent on the timing of sepsis induction, coinciding with the daily changes in TLR9 expression and function. These findings unveil a direct molecular link between the circadian and innate immune systems with important implications for immunoprophylaxis and immunotherapy.

Circadian Rhythms Influence the Severity of Sepsis in Mice via a TLR2-Dependent, Leukocyte-Intrinsic Mechanism.

Circadian rhythms coordinate an organism's activities and biological processes to the optimal time in the 24-h daylight cycle. We previously demonstrated that male C57BL/6 mice develop sepsis more rapidly when the disease is induced in the nighttime versus the daytime. In this report, we elucidate the mechanism of this diurnal difference. Sepsis was induced via cecal ligation and puncture (CLP) at zeitgeber time (ZT)-19 (2 am) or ZT-7 (2 pm). Like the males used in our prior study, female C57BL/6 mice had a worse outcome when CLP was induced at ZT-19 versus ZT-7, and these effects persisted when we pooled the data from both sexes. In contrast, mice with a mutated Period 2 (Per2) gene had a similar outcome when CLP was induced at ZT-19 versus ZT-7. Bone marrow chimeras reconstituted with C57BL/6 immune cells exhibited a worse outcome when sepsis was induced at ZT-19 versus ZT-7, whereas chimeras with Per2-mutated immune cells did not. Next, murine macrophages were subjected to serum shock to synchronize circadian rhythms and exposed to bacteria cultured from the mouse cecum at 4-h intervals for 48 h. We observed that IL-6 production oscillated with a 24-h period in C57BL/6 cells exposed to cecal bacteria. Interestingly, we observed a similar pattern when cells were exposed to the TLR2 agonist lipoteichoic acid. Furthermore, TLR2-knockout mice exhibited a similar sepsis phenotype when CLP was induced at ZT-19 versus ZT-7. Together, these data suggest that circadian rhythms in immune cells mediate diurnal variations in murine sepsis severity via a TLR2-dependent mechanism.

Increased Levels of Macrophage Inflammatory Proteins Result in Resistance to R5-Tropic HIV-1 in a Subset of Elite Controllers.

UNLABELLED: Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4(+) T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4(+) T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1α (MIP-1α), CCL3 and CCL3L1, were found to be upregulated. The MIP-1α, MIP-1ß, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1α and/or MIP-1ß at the protein level. The supernatant from resistant EC cells contained MIP-1α and MIP-1ß and was sufficient to confer R5-tropic resistance to susceptible CD4(+) T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry. IMPORTANCE: HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4(+) T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals.

Immunity's fourth dimension: approaching the circadian-immune connection.

The circadian system ensures the generation and maintenance of self-sustained ~24-h rhythms in physiology that are linked to internal and environmental changes. In mammals, daily variations in light intensity and other cues are integrated by a hypothalamic master clock that conveys circadian information to peripheral molecular clocks that orchestrate physiology. Multiple immune parameters also vary throughout the day and disruption of circadian homeostasis is associated with immune-related disease. Here, we discuss the molecular links between the circadian and immune systems and examine their outputs and disease implications. Understanding the mechanisms that underlie circadian-immune crosstalk may prove valuable for devising novel prophylactic and therapeutic interventions.

MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.

Common Variables That Influence Sepsis Mortality in Mice.

Introduction: Sepsis is characterized by a dysregulated host immune response to infection, leading to organ dysfunction and a high risk of death. The cecal ligation and puncture (CLP) mouse model is commonly used to study sepsis, but animal mortality rates vary between different studies. Technical factors and animal characteristics may affect this model in unanticipated ways, and if unaccounted for, may lead to serious biases in study findings. We sought to evaluate whether mouse sex, age, weight, surgeon, season of experiments, and timing of antibiotic administration influenced mortality in the CLP model. Methods: We created a comprehensive dataset of C57BL/6J mice that had undergone CLP surgery within our lab during years 2015-2020 from published and unpublished studies. The primary outcome was defined as the time from sepsis induction to death or termination of study (14 days). The Log rank test and Cox regression models were used to analyze the dataset. The study included 119 mice, of which 43% were female, with an average age of 12.6 weeks, an average weight of 25.3 g. 38 (32%) of the animals died. Results: In the unadjusted analyses, experiments performed in the summer and higher weight predicted a higher risk of mortality. In the stratified Cox model by sex, summer season (adjusted hazard ratio [aHR]=5.61, p=0.004) and delayed antibiotic administration (aHR=1.46, p=0.029) were associated with mortality in males, whereas higher weight (aHR=1.52, p=0.005) significantly affected mortality in females. In addition, delayed antibiotic administration (HR=1.42, p=0.025) was associated with mortality in the non-summer seasons, but not in the summer season. Discussion: In conclusion, some factors specific to sex and season have a significant influence on sepsis mortality in the CLP model. Consideration of these factors along with appropriate group matching or adjusted analysis is critical to minimize variability beyond the experimental conditions within a study.

GOODNIGHT, SLEEP TIGHT, DON'T LET THE MICROBES BITE: A REVIEW OF SLEEP AND ITS EFFECTS ON SEPSIS AND INFLAMMATION.

ABSTRACT: Sleep is a restorative biological process that is crucial for health and homeostasis. However, patient sleep is frequently interrupted in the hospital environment, particularly within the intensive care unit. Suboptimal sleep may alter the immune response and make patients more vulnerable to infection and sepsis. In addition, hospitalized patients with sepsis experience altered sleep relative to patients without infectious disease, suggesting a bidirectional interplay. Preclinical studies have generated complementary findings, and together, these studies have expanded our mechanistic understanding. This review article summarizes clinical and preclinical studies describing how sleep affects inflammation and the host's susceptibility to infection. We also highlight potential strategies to reverse the detrimental effects of sleep interruption in the intensive care unit.

TLR9 and IRF3 cooperate to induce a systemic inflammatory response in mice injected with liposome:DNA.

Liposome:DNA is a promising gene therapy vector. However, this vector can elicit a systemic inflammatory response syndrome (SIRS). Prior reports indicate that liposome:DNA vectors activate Toll-like receptor (TLR)9. We hypothesized that liposome:DNA vectors also activate the cytosolic DNA-sensing pathway, which signals via interferon (IFN) regulatory factor (IRF)3. To test this, we treated dendritic cells (DCs) with liposome:DNA in vitro and found that IRF3 was phosphorylated independent of TLR9. To test the contribution of this pathway in vivo, we injected a liposome:DNA vector into wild-type (WT), TLR9-knockout (KO), IRF3-KO, and TLR9-IRF3-double-KO (DKO) mice. WT mice exhibited a systemic inflammatory response, evidenced by elevations in serum cytokines, serum enzyme changes indicating organ damage, hypothermia, and mortality. The cytokine response was reduced in TLR9-KO, IRF3-KO, and TLR9-IRF3-DKO mice and all three groups survived. We found that IFN-gamma-KO mice that receive liposome:DNA had a reduced cytokine response and 100% survival. CD11c(+) and NK1.1(+) cells produced IFN-gamma and depleting CD11c(+) cells reduced the cytokine response in mice injected with liposome:DNA. These findings may facilitate the development of immunologically inert gene therapy vectors and may provide general insight into the mechanisms of SIRS.

Aged IRF3-KO Mice are Protected from Sepsis.

PURPOSE: Sepsis is a leading cause of hospital admissions and deaths. Older adults (>65 years) are particularly susceptible to sepsis and experience higher morbidity and mortality rates than younger people. We previously showed that interferon regulatory factor 3 (IRF3) contributes to sepsis pathogenesis in young mice subject to cecal ligation and puncture (CLP). In this study, we investigated if IRF3 contributes to sepsis in the context of aging. METHODS: Sepsis was induced in aged wild-type (WT) and IRF3-knock-out (KO) mice, using a clinically-relevant CLP-sepsis model including fluids and antibiotics. Animal survival, disease score and hypothermia were evaluated as indicators of sepsis pathogenesis. Serum cytokines and serum enzymes indicative of organ damage were also measured. RESULTS: Aged WT mice were highly susceptible to sepsis (90% mortality). In comparison, aged IRF3-KO mice were significantly protected (20% mortality). Aged IRF3-KO mice showed a lower disease score and reduced hypothermia following CLP, compared to WT mice. Serum cytokines interleukin (IL)-6, IL-12/23p40 and macrophage chemoattractant protein (MCP)-1, and creatinine kinase (CK) were lower in aged IRF3-KO septic mice compared to WT counterparts. Aged male mice were found to be more susceptible to sepsis compared to females. Female mice, however, produced higher levels of serum cytokines and CK. CONCLUSION: These results demonstrate that IRF3 plays a detrimental role in sepsis in aged mice and highlight the impact of biological sex.

Creation of BLT Humanized Mice for Sepsis Studies.

Mice are a suitable animal model for sepsis studies because they recapitulate many aspects of the pathophysiology observed in septic human patients. It is ethically preferable to use mice for research over higher sentient species, when scientifically appropriate. Mice are also advantageous for research due to their small size, modest housing needs, the availability of genetically modified strains, and the broad range of reagents available for scientific assays on this species. Nevertheless, there are some intrinsic differences between mice and humans that should be recognized when considering the translational potential of sepsis therapies. It is often wise to complement traditional mouse studies with animal models that exhibit even greater similarity to humans, and in particular, models that better recapitulate the human immune response. Humanized mice are a promising tool to bridge this interspecies research gap. Herein, we provide a protocol to generate BLT humanized mice and describe their sepsis phenotype after cecal ligation and puncture (CLP).

Detection of Blood Cell Surface Biomarkers in Septic Mice.

Sepsis arises when an infection induces a dysregulated immune response, resulting in organ damage. New methods are urgently needed to diagnose patients in the early stages of sepsis, and identify patients with a poor disease prognosis. One promising approach is to identify the rapid changes in cell surface antigens (biomarkers) that occur during sepsis, as a consequence of leukocyte mobilization and activation. This chapter describes the method for staining whole blood with fluorescently conjugated antibodies that detect cell surface biomarkers, and performing flow cytometry analysis to quantify biomarker-positive cells. Our protocol is designed to detect blood cell surface biomarkers in septic mice, but could also be applied to study potential biomarkers in blood obtained from human patients with sepsis and other medical conditions.

Large Peritoneal Macrophages and Transitional Premonocytes Promote Survival during Abdominal Sepsis.

Monocytes and macrophages are early sentinels of infection. The peritoneum contains two resident populations: large and small peritoneal macrophages (LPMs and SPMs). While LPMs self-renew, circulating monocytes enter the peritoneum and differentiate into SPMs. We lack information on the dynamics of monocyte-macrophage trafficking during abdominal sepsis, reflecting an important knowledge gap. In this study, we characterize the presence of LPMs, SPMs, and monocytes in the peritoneum of mice following cecal ligation and puncture (CLP)-induced sepsis and sham surgery. LPMs rapidly disappeared from the peritoneum and were scarce at 18-66 h after CLP or sham surgery. By 14 d, LPMs returned for sham mice, but they remained scarce in CLP mice. Depletion of LPMs from the peritoneum of CD11b-DTR mice greatly increased animal mortality. These data imply that LPMs are critical for sepsis survival. Monocytes rapidly infiltrated the peritoneum and were abundant at 18-66 h after CLP or sham surgery. Surprisingly, SPMs only increased at 14 d post-CLP. Therefore, monocytes may defend hosts from acute sepsis mortality without generating SPMs. More monocytes were present in mice predicted to survive sepsis versus mice predicted to die. However, altering monocyte numbers via CCR2 deficiency or adoptive transfer did not significantly affect animal survival. We reasoned that animals destined to survive sepsis may exhibit a different monocyte phenotype, rather than merely enhanced numbers. Indeed, mice predicted to survive possessed more CD31+, CXCR4hi transitional premonocytes in their abdomen. Inhibition of CXCL12-CXCR4 signaling via AMD3100 exacerbated sepsis. These data imply that recruitment of transitional premonocytes to the abdomen promotes sepsis survival.

Evaluating the Timeliness and Specificity of CD69, CD64, and CD25 as Biomarkers of Sepsis in Mice.

ABSTRACT: Sepsis occurs when an infection induces a dysregulated immune response, and is most commonly bacterial in origin. This condition requires rapid treatment for successful patient outcomes. However, the current method to confirm infection (blood culture) requires up to 48 h for a positive result and many true cases remain culture-negative. Therefore, new diagnostic tests are urgently needed. Recent clinical studies suggest that CD69, CD64, and CD25 may serve as useful biomarkers of sepsis. In this study, we evaluated the cecal ligation and puncture and cecal slurry mouse models as tools to study these biomarkers in young and aged mice, and elucidate the timeliness and specificity of sepsis diagnosis. Fluorescence-activated cell sorting analysis revealed that all three biomarkers were elevated on blood leukocytes during sepsis. CD69 was specifically upregulated during sepsis, while CD64 and CD25 were also transiently upregulated in response to sham surgery. The optimal biomarker, or combination of biomarkers, depended on the timing of detection, mouse age, and presence of surgery. CD69 demonstrated an excellent capacity to distinguish sepsis, and in some scenarios the diagnostic performance was enhanced by combining CD69 with CD64. We also analyzed biomarker expression levels on specific cell populations (lymphocytes, monocytes, and neutrophils) and determined the cell types that upregulate each biomarker. Elevations in blood biomarkers were also detected via microfluidic analyses; in this case CD64 distinguished septic mice from naive controls. Our results suggest that CD69 and CD64 are valuable biomarkers to rapidly detect sepsis, and that mouse models are useful to study and validate sepsis biomarkers.

Dual signaling of MyD88 and TRIF is critical for maximal TLR4-induced dendritic cell maturation.

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.

Aging impairs IFN regulatory factor 7 up-regulation in plasmacytoid dendritic cells during TLR9 activation.

Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-alpha in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with HSV-2, a viral pathogen that activates TLR9. We found that impaired IFN-alpha production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective up-regulation of IFN-regulatory factor 7, a key adaptor in the type I IFN pathway, as compared with younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-alpha defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.

Perfect timing: circadian rhythms, sleep, and immunity - an NIH workshop summary.

Recent discoveries demonstrate a critical role for circadian rhythms and sleep in immune system homeostasis. Both innate and adaptive immune responses - ranging from leukocyte mobilization, trafficking, and chemotaxis to cytokine release and T cell differentiation -are mediated in a time of day-dependent manner. The National Institutes of Health (NIH) recently sponsored an interdisciplinary workshop, "Sleep Insufficiency, Circadian Misalignment, and the Immune Response," to highlight new research linking sleep and circadian biology to immune function and to identify areas of high translational potential. This Review summarizes topics discussed and highlights immediate opportunities for delineating clinically relevant connections among biological rhythms, sleep, and immune regulation.

Methods to Study the Innate Immune Response to Sepsis.

This chapter describes techniques to measure the innate immune response in the mouse cecal ligation and puncture model of sepsis. The reader will learn how to perform retro-orbital bleeds to harvest serum from mice and learn how to perform peritoneal lavage to harvest cells and inflammatory mediators from this compartment. The enzyme-linked immunosorbent assay (ELISA) technique is described as a method to measure the levels of cytokines and chemokines in these fluids. Additionally, this chapter describes techniques to stain the cellular fraction of the peritoneal lavage with fluorescently labeled antibodies, and perform fluorescence activated cell sorting (FACS) to quantify macrophages and neutrophils in this compartment.