Early Pro-inflammatory Response Research Articles

MiRNA-Mediated Fine Regulation of TLR-Induced M1 Polarization.

Macrophage polarization to the M1 spectrum is induced by bacterial cell wall components through stimulation of Toll-like family (TLR) receptors. By orchestrating the expression of relevant mediators of the TLR cascade, as well as associated pathways and feedback loops, macrophage polarization is coordinated to ensure an appropriate immune response. This is central to the successful control of pathogens and the maintenance of health. Macrophage polarization is known to be modulated at both the transcriptional and post-transcriptional levels. In recent years, the miRNA-based post-transcriptional regulation of M1 polarization has received increasing attention from the scientific community. Comparative studies have shown that TLR stimulation alters the miRNA profile of macrophages and that macrophages from the M1 or the M2 spectrum differ in terms of miRNAs expressed. Simultaneously, miRNAs are considered critical post-transcriptional regulators of macrophage polarization. In particular, miRNAs are thought to play a regulatory role in the switch between the early proinflammatory response and the resolution phase. In this review, we will discuss the current state of knowledge on the complex interaction of transcriptional and post-transcriptional regulatory mechanisms that ultimately determine the functionality of macrophages.

NF-κB signaling deficiency in CD11c-expressing phagocytes mediates early inflammatory responses and enhances Mycobacterium tuberculosis control.

Early innate immune responses play an important role in determining the protective outcome of Mycobacterium tuberculosis (Mtb) infection. Nuclear factor kappa B (NF-κB) signaling in immune cells regulates the expression of key downstream effector molecules that mount early anti-mycobacterial responses. Using conditional knockout mice, we studied the effect of abrogation of NF-κB signaling in different myeloid cell types and its impact on Mtb infection. Our results show that absence of IKK2-mediated signaling in all myeloid cells resulted in increased susceptibility to Mtb infection. In contrast, absence of IKK2-mediated signaling specifically in CD11c+ myeloid cells induced early pro-inflammatory cytokine responses, enhanced the recruitment of myeloid cells and mediated early resistance to Mtb. Abrogation of IKK2 in MRP8-expressing neutrophils did not impact either disease pathology or Mtb control. Thus, we describe an early immunoregulatory role for NF-κB signaling in CD11c-expressing phagocytes, and a later protective role for NF-κB in LysM-expressing cells during Mtb infection.

CXCL17 is a proinflammatory chemokine and promotes neutrophil trafficking.

CXCL17, a novel member of the CXC chemokine class, has been implicated in several human pathologies, but its role in mediating immune response is not well understood. Characteristic features of immune response include resident macrophages orchestrating successive and structured recruitment of neutrophils and monocytes to the insult site. Here, we show that Cxcl17 knockout (KO) mice, compared with the littermate wild-type control mice, were significantly impaired in peritoneal neutrophil recruitment post-lipopolysaccharide (LPS) challenge. Further, the KO mice show dysregulated Cxcl1, Cxcr2, and interleukin-6 levels, all of which directly impact neutrophil recruitment. Importantly, the KO mice showed no difference in monocyte recruitment post-LPS challenge or in peritoneal macrophage levels in both unchallenged and LPS-challenged mice. We conclude that Cxcl17 is a proinflammatory chemokine and that it plays an important role in the early proinflammatory response by promoting neutrophil recruitment to the insult site.

Poster 238: Early Postoperative Increase of IL-1a in Synovial Fluid Correlates with Worse Short-Term Patient-Reported Outcomes after Cartilage Transplantation in the Knee

Objectives: The purpose of this study was to determine the longitudinal concentrations of several synovial fluid biomarkers after cartilage transplantation and potential correlations with patient reported outcomes. Methods: After institutional IRB approval, patients undergoing osteochondral allograft (OCA) transplantation or autochondrocyte implantation (ACI) were consented and enrolled into a prospective study involving intraoperative synovial fluid aspiration of the operative knee, as well as postoperative aspirations of the operative knee at standardized clinic follow-up timepoints: 2 weeks, 6 weeks, 6 months, and 1 year postoperatively. For this analysis, patients undergoing cartilage transplantation (OCA or ACI) with preoperative aspiration and at least one successful postoperative aspiration were included. Exclusion criteria for aspirations from the analysis included subsequent surgical procedure or articular injections on the index knee within 1 year of the transplant. The synovial fluid samples were analyzed for the following inflammatory biomarkers with Multiplex ELISA: CCL5, MMP-1, EGF, VEGF, IL1a, FGF2, CCL2, BMP2, and Aggrecan (ACAN). The samples were digested 1:1 with 3mg/mL hyaluronidase prior to execution of the assay in accordance with the manufacturer’s protocol. A minimum of 3uL was required for analysis. Patients prospectively completed Knee Injury and Osteoarthritis Outcome Score (KOOS), including Activities of Daily Living (ADL), Pain, Quality of Life (QoL), Sports, Symptoms, and Joint Replacement (JR) subscores, preoperatively and at 6 months and 1 year postoperatively. All statistical analyses were performed on RStudio and STATA. Non-parametric tests were utilized to avoid assumptions of normality. Skillings-Mack tests were utilized as a non-parametric alternative for a repeated-measures ANOVA in the setting of sporadic missing values (i.e., dry aspirations). Post-hoc pairwise Wilcoxon ranksum tests with Bonferroni corrections for multiple comparisons were performed for significant tests. Univariate linear regressions were performed to determine correlations between cytokine changes and patient-reported outcomes. A minimum of 10 observations per predictor was considered sufficient for the analysis. All testing was two-sided, and significance was set at p<0.05. Results: A total of 63 aspirations from 19 patients were evaluated. Postoperatively, successful aspirations were able to be performed on 13 patients at 2 weeks (68%), 17 patients at 6 weeks (89%), 9 patients at 6 months (47%), and 5 patients at 1 year (26%) (Table 1). One patient underwent a subsequent procedure on the index knee (lysis of adhesions) 10 weeks after cartilage transplantation, thereby excluding their 6 month and 1-year aspirations from analysis. Across all timepoints, there were significant differences in MMP-1 (p=0.003), ACAN (p<0.001), and FGF2 (p=0.026). Specifically, ACAN was significantly elevated at 2 (p<0.001) and 6 weeks (p<0.001), and MMP- 1 was the greatest at 6 weeks relative to transplant (p<0.001), 6 months (p=0.01), and 1 year (p=0.04). In contrast, FGF2 was significantly lower at 2 weeks compared to transplant levels (p=0.02). There were no significant differences across timepoints for CCL5 (p=0.88), VEGF (p=0.068), CCL2 (p=0.071), BMP2 (p=0.067), IL-1a (p=0.13), or EGF (p=0.01, p>0.05 for all pairwise comparisons after Bonferroni correction) (Figure 1). A subanalysis was performed for cytokines which demonstrated at least 5 “pro-inflammatory” responses (positive fold-change) and at least 5 “anti-inflammatory” responses (negative fold-change), with a minimum of 10 observations with associated patient-reported outcomes at 6 and/or 12 months postoperatively. From the 2-week aspiration samples, CCL5 (5 increase/7 decrease), IL-1a (5/7), and CCL2 (6/7) met the inclusion criteria. Increase in IL-1a demonstrated significant linear associations with worse postoperative KOOS ADL (p=0.048), Pain (p=0.01), Quality of Life (p=0.01), and Symptoms (p=0.01) scores at 6 months (Table 2, Figure 2). Similarly, an increase in CCL-2 postoperatively was associated with worse KOOS ADL (p=0.04) and Pain (p=0.04) scores at 6 months. No significant linear associations between cytokine changes at 6 weeks and later PROMs were identified. Conclusions: Following cartilage transplantation of the knee, the synovial fluid profile may demonstrate early postoperative increases in MMP-1 and ACAN and a decrease in FGF-2. An early pro-inflammatory response with IL-1a within the first two postoperative weeks may be linearly associated with worse short-term patient-reported outcomes compared to an early decrease. [Table: see text] [Table: see text]

Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.

The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.

Multiplexed trackable intratumor microdosing of the investigational STING agonist TAK-676 alone and in combination in the native tumor microenvironment of patients with head and neck cancer: A phase 0 trial.

2579 Background: TAK-676 is a novel synthetic STING agonist developed to induce an inflammatory state through release of interferon (IFN), production of proinflammatory chemokines and cytokines, and activation of innate and adaptive immune cells in the tumor microenvironment (TME). To gain early mechanistic insights on the effects of TAK-676 in the native TME alone and in combination with standard-of-care chemotherapies, we completed a Phase 0 intratumoral microdosing trial in head and neck squamous cell carcinoma patients using the Comparative In Vivo Oncology (CIVO) platform. CIVO enables direct evaluation of biomarker response in trackable, distinct regions of drug exposure upon resection. These studies were designed to identify rational combinations that could be evaluated in future clinical trials with therapeutic intent. Methods: This IRB-approved study was conducted at four centers within the United States. Fifteen subjects provided Informed Consent and enrolled in the trial. Microdoses (≤1/100th systemic dose) of TAK-676, carboplatin, 5-FU, paclitaxel, and combinations thereof, were simultaneously injected via CIVO to eligible patients presenting with a surface-accessible primary or metastatic lesion ≥ 2 cm and planned surgical resection of the target lesion at 24 (n=8) or 72–96 (n=7) hours. Injection sites were identified via co-injection of fluorescent tracking markers (CIVO GLO). Multiplexed ISH, IHC, and spatial molecular profiling via GeoMx and CosMx were used to resolve molecular responses at each injection site. Patients were monitored for adverse events (AEs) for 28 days following injection. Results: TAK-676 exposure was associated with increased IFN signaling in all samples analyzed at the 24-hour cohort. This was accompanied by increased expression of pro-inflammatory cytokines and chemokines, “M1-like” macrophages, NK cells, and CD8+ T cells. Spatial profiling with GeoMx and CosMx revealed an IFN signature and elevated pro-inflammatory markers across multiple cell types throughout the TME. In addition, exposure to TAK-676 plus chemotherapy resulted in localized enhancement of apoptosis vs. chemotherapy alone across multiple tumors. The responses observed at 24 hours were transient and not detected in the 72–96-hour cohort. No AEs related to injection procedure or contents were reported. Conclusions: Intratumor microdosing via CIVO of TAK-676 alone and in combination with chemotherapy was shown to promote an early pro-inflammatory response, providing evidence of an on-target mechanism of action. The absence of effect at the late timepoints could be due to pharmacokinetics of drug exposure or compensatory signaling within the TME. In this Phase 0 clinical trial we safely demonstrated precise, mechanistic effects of TAK-676 on the native TME of intact human tumors. Clinical trial information: NCT04541108 .

The mechanism of microglia-mediated immune inflammation in ischemic stroke and the role of natural botanical components in regulating microglia: A review.

Ischemic stroke (IS) is one of the most fatal diseases. Neuroimmunity, inflammation, and oxidative stress play important roles in various complex mechanisms of IS. In particular, the early proinflammatory response resulting from the overactivation of resident microglia and the infiltration of circulating monocytes and macrophages in the brain after cerebral ischemia leads to secondary brain injury. Microglia are innate immune cells in the brain that constantly monitor the brain microenvironment under normal conditions. Once ischemia occurs, microglia are activated to produce dual effects of neurotoxicity and neuroprotection, and the balance of the two effects determines the fate of damaged neurons. The activation of microglia is defined as the classical activation (M1 type) or alternative activation (M2 type). M1 type microglia secrete pro-inflammatory cytokines and neurotoxic mediators to exacerbate neuronal damage, while M2 type microglia promote a repairing anti-inflammatory response. Fine regulation of M1/M2 microglial activation to minimize damage and maximize protection has important therapeutic value. This review focuses on the interaction between M1/M2 microglia and other immune cells involved in the regulation of IS phenotypic characteristics, and the mechanism of natural plant components regulating microglia after IS, providing novel candidate drugs for regulating microglial balance and IS drug development.

Activation of Early Proinflammatory Responses by TBEV NS1 Varies between the Strains of Various Subtypes.

Tick-borne encephalitis (TBE) is an emerging zoonosis that may cause long-term neurological sequelae or even death. Thus, there is a growing interest in understanding the factors of TBE pathogenesis. Viral genetic determinants may greatly affect the severity and consequences of TBE. In this study, nonstructural protein 1 (NS1) of the tick-borne encephalitis virus (TBEV) was tested as such a determinant. NS1s of three strains with similar neuroinvasiveness belonging to the European, Siberian and Far-Eastern subtypes of TBEV were studied. Transfection of mouse cells with plasmids encoding NS1 of the three TBEV subtypes led to different levels of NS1 protein accumulation in and secretion from the cells. NS1s of TBEV were able to trigger cytokine production either in isolated mouse splenocytes or in mice after delivery of NS1 encoding plasmids. The profile and dynamics of TNF-α, IL-6, IL-10 and IFN-γ differed between the strains. These results demonstrated the involvement of TBEV NS1 in triggering an immune response and indicated the diversity of NS1 as one of the genetic factors of TBEV pathogenicity.

Parietal composition of Lichtheimia corymbifera: differences between spore and germ tube stages and host-pathogen interactions.

The molecular composition and structural organisation of the cell wall of filamentous fungi underlie the ability of the host to identify them as pathogens. Although the organisation of the fungal cell wall, composed of 90% polysaccharides, is similar from one fungus to another, small variations condition their ability to trigger pattern recognition receptors. Because the incidence of mucormycosis, an emerging life-threatening infection caused by the species of the order Mucorales is increasing worldwide, the precise composition of the cell wall of two strains of Lichtheimia corymbifera was investigated in the early growth stages of germination (spores and germ-tubes) using trimethylsilylation and confocal microscopy. This study also characterises the response of THP-1 cells to Mucorales. The study identified the presence of uncommon monosaccharides (fucose, galactose and glucuronic acid) whose respective proportions vary according to the germination stage, revealing early parietal reorganisation. Immunofluorescence studies confirmed the exposure of β-glucan on the surface of swollen spores and germ-tubes. Both spores and germ-tubes of L. corymbifera promoted an early and strong pro-inflammatory response, through TLR-2. Our results show the singularity of the cell wall of the order Mucorales, opening perspectives for the development of specific diagnostic biomarkers.

Conventional and Pro-Inflammatory Pathways of Fibrinolytic Activation in Non-Traumatic Hyperfibrinolysis.

Hyperfibrinolysis (HF) frequently occurs after severe systemic hypoperfusion during major trauma and out-of-hospital cardiac arrest (OHCA). In trauma-induced HF, hypoperfusion, the activation of protein C (APC), and the release of tissue plasminogen activator (t-PA) have been identified as the driving elements of premature clot breakdown. The APC pathway also plays a role in inflammatory responses such as neutrophil extracellular trap formation (NETosis), which might contribute to lysis through cleavage of fibrin by neutrophil elastases. We investigated whether the APC and the plasminogen pathway were general drivers of HF, even in the absence of a traumatic incident. Additionally, we were interested in inflammatory activation such as the presence of NETs as potential contributing factors to HF. A total of 41 patients with OHCA were assigned to a HF and a non-HF group based on maximum lysis (ML) in thromboelastometry. Thrombin-antithrombin (TAT)-complex, soluble thrombomodulin (sTM), APC-PC inhibitor complex, t-PA, PAI-1, t-PA-PAI-1 complex, plasmin-antiplasmin (PAP), d-dimers, neutrophil elastase, histonylated DNA (hDNA) fragments, and interleukin-6 were assessed via immunoassays in the HF group vs. non-HF. APC-PC inhibitor complex is significantly higher in HF patients. Antigen levels of t-PA and PAI-1 do not differ between groups. However, t-PA activity is significantly higher and t-PA-PAI-1 complex significantly lower in the HF group. Consistent with these results, PAP and d-dimers are significantly elevated in HF. HDNA fragments and neutrophil elastase are not elevated in HF patients, but show a high level of correlation, suggesting NETosis occurs in OHCA as part of inflammatory activation and cellular decay. Just as in trauma, hypoperfusion, the activation of protein C, and the initiation of the plasminogen pathway of fibrinolysis manifest themselves in the HF of cardiac arrest. Despite features of NETosis being detectable in OHCA patients, early pro-inflammatory responses do not appear be associated with HF in cardiac arrest.

41 Peripheral Mononuclear Cell Expression of IL-10, IL-12, ARG1, & NOS2 Correlates with Outcomes After Burn

IntroductionIn the burned patient, clinical outcomes are inextricably linked with immune function. Patients are subject to an early pro-inflammatory response and a subsequent compensatory anti-inflammatory response syndrome. This dysregulation can lead to infection, multiple organ dysfunction syndrome, and death. Despite continuing efforts, a profile of immune gene expression from burn patients that can be transformed into an “immune suppression index”, which accurately reflects the underlying degree of immune insult and significantly correlates with the degree compromise, has yet to be developed. The development of such an approach that can predict graft failure, susceptibility to infection, length-of-stay in the hospital, and/or that can inform a provider on how to better manage the timing of surgical interventions would be transformative.ObjectiveTo determine a set of peripheral biomarkers that correlates with clinical outcomes of burn patients.MethodsThis observational study enrolled two participant cohorts within a single burn center. Initial unbiased analysis compared 23 burn patients and 6 healthy controls. Confirmatory outcomes analysis was performed in 109 burn patients and 19 healthy controls. We employed multiplex gene expression analysis to identify differential peripheral blood mononuclear cells (PBMC) immune gene expression. qPCR was used to validate these findings, identify, and model associations with outcomes.ResultsWe identified 149 genes with a significant difference in expression within PBMCs from burn patients compared to controls (Figure 1a). Pathway analysis identified pathways related to IL-10 and inducible nitric oxide synthase (iNOS) signaling (Figure 1b). qPCR analysis of IL-10, IL-12, arginase-1 (ARG1), and iNOS demonstrated that burn injury was associated with increased expression of ARG1 and IL-10, and decreased expression of NOS2 and IL-12. Burn severity, acute lung injury (ALI), development of infection, failure of skin autograft, and mortality significantly correlated with expression of one or more of these genes. Ratios of IL-10/IL-12, ARG1/NOS2 and (ARG1+IL-10)/(NOS2+IL-12) transcript levels further improved the correlation with outcomes. A multivariate regression model, adjusting for confounders, demonstrated that (ARG1+IL-10)/(NOS2+IL-12) significantly correlated with burn severity and development of ALI (Table 1).ConclusionsWe present a robust model to predict patient outcomes early after burn injury using non-invasive methods, allowing early identification of underlying immune dysfunction.

Antifungals influence the immune-related transcriptomic landscape of human monocytes after Aspergillus fumigatus infection

The heterogeneity of clinical responses to antifungals in aspergillosis is partially understood. We hypothesized that besides direct antifungal effects, these discrepancies may be related to different immunomodulatory profiles. Human THP-1 monocytes were coincubated in vitro with Aspergillus fumigatus and variable concentrations of voriconazole (0.5, 1 and 2 mg/l), caspofungin (1 and 2 mg/l), amphotericin B deoxycholate (0.25, 0.5 and 1 mg/l) and liposomal amphotericin B (1, 2 and 3 mg/l). After 6 h of coincubation, total cellular RNA was extracted, converted into cDNA, and transcription of 84 genes involved in antifungal immunity was measured through RT-qPCR. The presence of A. fumigatus was the main driver of the global immune-related transcriptomic response. After Aspergillus infection, thirty genes were upregulated, while 19 genes were downregulated. Discrepancies across antifungals were also evident; voriconazole-containing conditions showed similar reaction to natural infection, while the use of liposomal Amphotericin B significantly decreased the inflammatory response. Chemokines (notably CCL20 and CXCL2) and pro-inflammatory cytokines (IL1A, IL1B, IL23, G-CSF) exhibited the most pronounced differences across antifungals. Pattern recognition receptors and adaptor protein transcription were minimally affected. Protein–protein-interaction network analysis showed that IL23A played a dominant role in upregulated genes. Pathway enrichment analysis indicated that cytokine-cytokine receptor integration, TNF signaling pathways and Toll-like receptor pathways were highly involved. This exploratory study confirms the heterogeneous immunomodulatory role of antifungals. Overall, voriconazole appears to maintain an early pro-inflammatory response seen in natural infection. Assessment of immunomodulatory response with clinical response may provide a better rationale for differences observed across antifungals.

Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis.

Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of co-expressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogen-related gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-γ signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPARγ in synovium from inflamed joints treated with BMNC, and equivalent IL-1β staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-γ signaling enhancement for the treatment of arthritic conditions.

Early Transcriptomic Response to Burn Injury: Severe Burns Are Associated With Immune Pathway Shutdown.

Burn injury induces a systemic hyperinflammatory response with detrimental side effects. Studies have described the biochemical changes induced by severe burns, but the transcriptome response is not well characterized. The goal of this work is to characterize the blood transcriptome after burn injury. Burn patients presenting to a regional center between 2012 and 2017 were prospectively enrolled. Blood was collected on admission and at predetermined time points (hours 2, 4, 8, 12, and 24). RNA was isolated and transcript levels were measured with a gene expression microarray. To identify differentially regulated genes (false-discovery rate ≤0.1) by burn injury severity, patients were grouped by TBSA above or below 20% and statistically enriched pathways were identified. Sixty-eight patients were analyzed, most patients were male with a median age of 41 (interquartile range, 30.5-58.5) years, and TBSA of 20% (11%-34%). Thirty-five patients had % TBSA injury ≥20%, and this group experienced greater mortality (26% vs 3%, P = .008). Comparative analysis of genes from patients with </≥20% TBSA revealed 1505, 613, 380, 63, 1357, and 954 differentially expressed genes at hours 0, 2, 4, 8, 12, and 24, respectively. Pathway analysis revealed an initial up-regulation in several immune/inflammatory pathways within the ≥20% TBSA groups followed by shutdown. Severe burn injury is associated with an early proinflammatory immune response followed by shutdown of these pathways. Examination of the immunoinflammatory response to burn injury through differential gene regulation and associated immune pathways by injury severity may identify mechanistic targets for future intervention.

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia.

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early pro-inflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia.

An early proinflammatory transcriptional response to tau pathology is age-specific and foreshadows reduced tau burden.

Age is one of the strongest risk factors for the development of neurodegenerative diseases, the majority of which involve misfolded protein aggregates in the brain. These protein aggregates are thought to drive pathology and are attractive targets for the development of new therapies. However, it is unclear how age influences the onset of pathology and the accompanying molecular response. To address this knowledge gap, we used a model of seeded tau pathology to profile the transcriptomic changes in 3 and 12 month old mice in response to developing tau hyperphosphorylation and aggregation. First, we found the burden of hyperphosphorylated tau pathology in mice injected at 12 months of age was moderately reduced compared to animals injected at 3 months. On a molecular level, we found an inflammation‐related subset of genes, including C3 and the disease‐associated microglia genes Ctsd, Cst7, and Clec7a, were more expressed early in disease in 12 but not 3 month old mice. These findings provide evidence of an early, age‐specific response to tau pathology, which could serve as a marker for the severity of downstream pathology.

CARD9 Deficiency Increases Hippocampal Injury Following Acute Neurotropic Picornavirus Infection but Does Not Affect Pathogen Elimination

Neurotropic viruses target the brain and contribute to neurologic diseases. Caspase recruitment domain containing family member 9 (CARD9) controls protective immunity in a variety of infectious disorders. To investigate the effect of CARD9 in neurotropic virus infection, CARD9−/− and corresponding C57BL/6 wild-type control mice were infected with Theiler’s murine encephalomyelitis virus (TMEV). Brain tissue was analyzed by histology, immunohistochemistry and molecular analyses, and spleens by flow cytometry. To determine the impact of CARD9 deficiency on T cell responses in vitro, antigen presentation assays were utilized. Genetic ablation of CARD9 enhanced early pro-inflammatory cytokine responses and accelerated infiltration of T and B cells in the brain, together with a transient increase in TMEV-infected cells in the hippocampus. CARD9−/− mice showed an increased loss of neuronal nuclear protein+ mature neurons and doublecortin+ neuronal precursor cells and an increase in β-amyloid precursor protein+ damaged axons in the hippocampus. No effect of CARD9 deficiency was found on the initiation of CD8+ T cell responses by flow cytometry and co-culture experiments using virus-exposed dendritic cells or microglia-enriched glial cell mixtures, respectively. The present study indicates that CARD9 is dispensable for the initiation of early antiviral responses and TMEV elimination but may contribute to the modulation of neuroinflammation, thereby reducing hippocampal injury following neurotropic virus infection.

Decellularization protocol-dependent damage-associated molecular patterns in rat uterus scaffolds differentially affect the immune response after transplantation.

Scaffolds derived from decellularized tissue possess many advantages for bioengineering applications, including for novel infertility treatments. However, the decellularization process results in allogenic-independent damage-associated molecular patterns (DAMPs). This field is poorly studied, in particular for uterus bioengineering applications. An increased knowledge concerning the immune system activation after transplantation of decellularized tissue will enable safer construct development and thereby accelerate translation from research to clinic. We therefore transplanted rat uterus scaffolds produced by three different decellularization protocols based on Triton X-100 (P1 and P2) or sodium deoxycholate (P3) in a syngeneic animal model and assessed the immune response towards DAMPs exposed by the decellularization process. Biopsies were retrieved on day 5, 15, and 30 post transplantation and immunohistochemistry-stained CD45+ (leucocytes), CD4+ (T-cells), CD8a+ (cytotoxic T-cells), CD22+ (B-cells), NCR1+ (NK-cells), CD68+ (pan-macrophages), and CD163+ (M2 macrophages) cells within the grafts were quantified. The gene expression for interferon γ, interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF) eotaxin-2, RANTES, MCP-1, MIP-1α, MIP-3α, IL-8 were also measured. Scaffolds from P1 induced a rapid cell infiltration after transplantation, presumably induced by DNA-based DAMPs. However, this response was only transient. Protocol 3 derived scaffolds induced an early pro-inflammatory cytokine response at the transcript level which remained high throughout the study. This response may be caused by the stronger decellularization detergent that could expose more extracellular matrix-related DAMPs. However, earlier proteomics analysis also identified significantly more abundant heat shook proteins-related DAMPs in this scaffold type. Protocol 2 caused the least immunogenic scaffolds and should thus be the future focus for in vivo uterus bioengineering applications.

A probiotic and mixed-enzymes combination reduces the inflammatory response, faecal shedding and systemic spread of Campylobacter jejuni in broilers

Recent research has questioned the notion that Campylobacter jejuni is a harmless resident of the avian gastrointestinal tract (GIT). The following trial examined the effect of dietary supplementation with a mixture of enzymes and Bacillus-based probiotics on growth performance, C. jejuni colonisation, GIT immune responses, faecal shedding and extra-intestinal spread in broilers. Fifty-eight, Ross 308 d-old broilers were randomly assigned to one of four treatments, giving 14 or 15 birds/pen. Birds were given nutritionally complete, complex, phased diets unsupplemented (two treatments) or supplemented (two treatments) with a multi-enzyme containing 2,000 U/kg xylanase, 200 U/kg amylase and 4,000 U/kg protease, and 75,000 cfu/g of a combination of three strains of Bacillus amyloliquefaciens. One control and one supplemented diet group contained birds orally challenged with 105 cfu C. jejuni strain M1 on d 21 of age. Among challenged birds, the mixed-enzymes and probiotic combination numerically reduced faecal C. jejuni shedding (-98% vs challenged control) three days-post-infection (d.p.i.), and at 7 d.p.i. numerically reduced C. jejuni colonisation of the ileal mucosa (-1000-fold vs control) and totally inhibited systemic spread of C. jejuni to the liver, compared to the control (P&lt;0.05). It suppressed early pro-inflammatory chemokine response seen in the ileum, caecum and caecal tonsil tissues (at 3 d.p.i.) in challenged control birds (-18 to -46-fold; P&lt;0.05) and altered expression of pro-inflammatory (IL-1β, IL-6, IL-17A and IL-17F) and regulatory (IL-10 and TGF-β) cytokines. The data demonstrated inhibition of a sustained pro-inflammatory response to C. jejuni infection and improved intestinal barrier integrity in supplemented birds. This highlighted the importance of looking beyond simple measurements of feed conversion and body weight gain when seeking to understand the effects and mode of action of poultry dietary interventions. Achieving a favourable balance between the gut, immune function, microbiome and nutrition should be the goal for achieving good gastrointestinal health and optimal performance.

126 Early Transcriptomic Response to Burn injury: Prolonged Inflammatory Response is Associated with Mortality

Abstract Introduction Burn injuries are associated with high morbidity and mortality. Burn care has improved significantly in the last few decades with emphasis on early surgical management, improvements in local wound care, and specialized critical care. While survival rates are improving, mortality remains high in certain patient populations, including those with larger burns. Burn injury induces a systemic hyperinflammatory response with detrimental side effects. Prior studies have offered early insights into the biochemical changes that occur after severe burn injury. The underlying cellular response is still largely unknown. The goal of this work is to characterize the blood transcriptome of severe burn injury and compare this response between patients who live or subsequently die of their injuries. Methods Burn patients presenting to a regional center between 2012–2017 were prospectively enrolled. Blood was collected on admission and at predetermined timepoints (Hours 2, 4, 8, 12, 24) over the first 24 hours. mRNA was isolated and a transcriptomic microarray was used to measure global transcript levels over time. To identify differentially regulated genes (FDR≤0.1) by injury severity, patients were grouped by burn size (TBSA &amp;gt;20%) and mortality. Microarray data was analyzed using bioinformatics software and pathway analysis. Descriptive statistics were generated with Mann-Whitney, Chi-Square, and Fisher’s exact test as appropriate. Results Sixty-eight patients were included in this analysis, most patients were male with a median age of 41 (IQR, 30.5–58.5) years, and TBSA of 20% (IQR, 11–34%). Thirty-five patients suffered %TBSA injury &amp;gt;20%, and this group experienced greater mortality (26% vs. 3%, p=0.008). There were no significant differences in age, race, or gender. Comparative analysis of genes from patients with &amp;lt; /&amp;gt;20% TBSA revealed 1250, 444, 209, 20, 865, and 557 differentially regulated genes at hours 0, 2, 4, 8, 12 and 24 respectively. Pathway analysis reveals an initial upregulation in several immune/inflammatory pathways within the &amp;gt;20% TBSA groups between hours 0–2 followed by shutdown between hours 12–24. Immune pathways include Th17 activation pathway and natural killer cell signaling, inflammatory pathways include EIF2 signaling. These pathways remain upregulated in the group of patients with &amp;gt;20% TBSA who died. Conclusions Severe burn injury is associated with an early proinflammatory immune response followed by shutdown of these pathways. Burn patients who die show continued upregulation in the first 24 hours after injury in several proinflammatory pathways compared to those who live.