Impaired Neutrophil Recruitment Research Articles

CD5L as a promising biological therapeutic for treating sepsis

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L’s important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L-/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans.

Loss of AC10 Impairs Neutrophil Recruitment and Alters Bacterial Colonization in the Lung During Bacterial Pneumonia

Rationale: Pseudomonas aeruginosa is a gram-negative bacterium and a common cause of nosocomial pneumonia that can progress to acute respiratory distress syndrome (ARDS), characterized by hypoxia, pulmonary edema, elevated bicarbonate, and widespread inflammation. Neutrophil recruitment is a critical line of defense against P. aeruginosa; however, excessive, or dysregulated neutrophil infiltration can exacerbate tissue damage and contribute to the severity of ARDS. Recently, AC10 has been implicated in neutrophil transendothelial migration via CD99. Despite the growing recognition and significance of AC10 in various physiological processes, its role in neutrophil recruitment during ARDS remains poorly understood. Thus, herein we hypothesize that loss of AC10 attenuates neutrophil recruitment during pneumonia and increase bacterial colonization in the lung. Methods: Wild-type and AC10 knockout mice were intratracheally inoculated with increasing doses of P. aeruginosa. Twenty fours after infection, bronchoalveolar lavage fluid (BALF) was collected and the percent of PMNs in the BALF determined. In separate experiments, mice were intratracheally inoculated with P. aeruginosa for 6 or 24 hours and organs (lungs, kidney, liver, spleen and brain) harvested, homogenized and the homogenate cultured overnight on Pseudomonas isolation agar for quantification of colony forming units (CFUs). Results: Our data confirmed CD99 expression in rat pulmonary microvascular endothelial cells and whole lung of both wild type and AC10 knockout mice. While there was no difference in the percentage of PMNs in the BALF between male wild type and knockout animals at any of the bacterial doses tested, in the female AC10 knockout mice, there was a decrease in the percentage of cells PMNs in the BALF compared to female wild type litter mates at several doses tested (1x103 up to 1x105, p<0.05). At a higher dose of P. aeruginosa (2.5x106), there was no difference in bacterial clearance between male WT and AC10 knockout mice, but a trend toward an increase in the CFU harvested from lung tissue 6 hours post infection in female KO mice. Conclusion: Thus, CD99 is expressed in the pulmonary endothelium. Further, in female mice, AC10 plays a role in neutrophil recruitment into the airspace. Further studies are required to resolve whether AC10 plays a role in bacterial clearance in female mice in a model of P. aeruginosa pneumonia. HL121513. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell surface RNAs control neutrophil recruitment

RNAs localizing to the outer cell surface have been recently identified in mammalian cells, including RNAs with glycan modifications known as glycoRNAs. However, the functional significance of cell surface RNAs and their production are poorly known. We report that cell surface RNAs are critical for neutrophil recruitment and that the mammalian homologs of the sid-1 RNA transporter are required for glycoRNA expression. Cell surface RNAs can be readily detected in murine neutrophils, the elimination of which substantially impairs neutrophil recruitment to inflammatory sites invivo and reduces neutrophils' adhesion to and migration through endothelial cells. Neutrophil glycoRNAs are predominantly on cell surface, important for neutrophil-endothelial interactions, and can be recognized by P-selectin (Selp). Knockdown of the murine Sidt genes abolishes neutrophil glycoRNAs and functionally mimics the loss of cell surface RNAs. Our data demonstrate the biological importance of cell surface glycoRNAs and highlight a noncanonical dimension of RNA-mediated cellular functions.

Reduction in mRNA Expression of the Neutrophil Chemoattract Factor CXCL1 in Pseudomonas aeruginosa Treated Barth Syndrome B Lymphoblasts.

Barth Syndrome (BTHS) is a rare X-linked genetic disease caused by a mutation in the TAFAZZIN gene, which codes for the protein tafazzin involved in cardiolipin remodeling. Approximately 70% of patients with BTHS exhibit severe infections due to neutropenia. However, neutrophils from BTHS patients have been shown to exhibit normal phagocytosis and killing activity. B lymphocytes play a crucial role in the regulation of the immune system and, when activated, secrete cytokines known to attract neutrophils to sites of infection. We examined the expression of chemokine (C-X-C motif) ligand 1 (CXCL1), a known chemotactic for neutrophils, in Epstein-Barr virus transformed control and BTHS B lymphoblasts. Age-matched control and BTHS B lymphoblasts were incubated with Pseudomonas aeruginosa for 24 h and then cell viability, CD27+, CD24+, CD38+, CD138+ and PD1+ surface marker expression and CXCL1 mRNA expression determined. Cell viability was maintained in lymphoblasts incubated in a ratio of 50:1 bacteria:B cells. Surface marker expression was unaltered between control and BTHS B lymphoblasts. In contrast, CXCL1 mRNA expression was reduced approximately 70% (p < 0.05) in untreated BTHS B lymphoblasts compared to control and approximately 90% (p < 0.05) in bacterial treated BTHS B lymphoblasts compared to the control. Thus, naïve and bacterial-activated BTHS B lymphoblasts exhibit reduced mRNA expression of the neutrophil chemoattractant factor CXCL1. We suggest that impaired bacterial activation of B cells in some BTHS patients could influence neutrophil function via impairing neutrophil recruitment to sites of infection and this could potentially contribute to these infections.

Comparative single-cell transcriptome analysis informs early innate immune response in severe human burns

Abstract Proper wound healing requires a coordinated time-dependent balance of immune pathways to prevent irreversible systemic damage. Given that innate immune cells are first to mobilize to sites of injury, we hypothesize that neutrophils, macrophages, and their interactions are associated with burn severity. To test this, we employed systems immunology and bioinformatics approaches utilizing single-cell transcriptome (scRNA-Seq) in model organisms and large-scale bulk transcriptome of human burns. To this end, we generated the first to-date scRNA-Seq atlas of 6,671 neutrophils and 12,560 macrophages from a time-course zebrafish tail burn model. Our data unveiled transcriptionally distinct subsets that present different dynamics during the wound healing process. We identified the granularity of IL6-IL6R interactions between macrophages and neutrophils, which impair neutrophil recruitment to sterile thermal injury, making the area vulnerable to microbial invasion and subsequent infection. Furthermore, we generated a comprehensive cross-species reference of neutrophils and macrophages in injury models by integrating publicly available mice scRNA-Seq with our zebrafish data. This reference allows us to deconvolute bulk transcriptome data from different published human cohorts and discover signatures of early innate immune cells associated with burn severity in humans. Our discovery demonstrates the power of using comparative bioinformatics approach to better understand the innate immune system during wound healing. In the future, these advances will guide interventional genomics approaches to identify druggable pathways to optimize burn wound healing in human patients.

Fpr2 exacerbates Streptococcus suis-induced streptococcal toxic shock-like syndrome via attenuation of neutrophil recruitment.

The life-threatening disease streptococcal toxic shock-like syndrome (STSLS), caused by the bacterial pathogen Streptococcus suis (S. suis). Proinflammatory markers, bacterial load, granulocyte recruitment, and neutrophil extracellular traps (NETs) levels were monitored in wild-type (WT) and Fpr2-/- mice suffering from STSLS. LXA4 and AnxA1, anti-inflammatory mediators related to Fpr2, were used to identity a potential role of the Fpr2 in STSLS development. We also elucidated the function of Fpr2 at different infection sites by comparing the STSLS model with the S. suis-meningitis model. Compared with the WT mice, Fpr2-/- mice exhibited a reduced inflammatory response and bacterial load, and increased neutrophil recruitment. Pretreatment with AnxA1 or LXA4 impaired leukocyte recruitment and increased both bacterial load and inflammatory reactions in WT but not Fpr2-/- mice experiencing STSLS. These results indicated that Fpr2 impairs neutrophil recruitment during STSLS, and this impairment is enhanced by AnxA1 or LXA4. By comparing the functions of Fpr2 in different S. suis infection models, inflammation and NETs was found to hinder bacterial clearance in S. suis meningitis, and conversely accelerate bacterial clearance in STSLS. Therefore, interference with neutrophil recruitment could potentially be harnessed to develop new treatments for this infectious disease.

Staphylococcus aureus induces a muted host response in human blood that blunts the recruitment of neutrophils

Staphylococcus aureus is an opportunistic pathogen and chief among bloodstream-infecting bacteria. S. aureus produces an array of human-specific virulence factors that may contribute to immune suppression. Here, we defined the response of primary human phagocytes following infection with S. aureus using RNA-sequencing (RNA-Seq). We found that the overall transcriptional response to S. aureus was weak both in the number of genes and in the magnitude of response. Using an ex vivo bacteremia model with fresh human blood, we uncovered that infection with S. aureus resulted in the down-regulation of genes related to innate immune response and cytokine and chemokine signaling. This muted transcriptional response was conserved across diverse S. aureus clones but absent in blood exposed to heat-killed S. aureus or blood infected with the less virulent staphylococcal species Staphylococcus epidermidis. Notably, this signature was also present in patients with S. aureus bacteremia. We identified the master regulator S. aureus exoprotein expression (SaeRS) and the SaeRS-regulated pore-forming toxins as key mediators of the transcriptional suppression. The S. aureus-mediated suppression of chemokine and cytokine transcription was reflected by circulating protein levels in the plasma. Wild-type S. aureus elicited a soluble milieu that was restrictive in the recruitment of human neutrophils compared with strains lacking saeRS. Thus, S. aureus blunts the inflammatory response resulting in impaired neutrophil recruitment, which could promote the survival of the pathogen during invasive infection.

Collaborative Action of Microglia and Astrocytes Mediates Neutrophil Recruitment to the CNS to Defend against Escherichia coli K1 Infection.

Escherichia coli K1 is a leading cause of neonatal bacterial meningitis. Recruitment of neutrophils to the central nervous system (CNS) via local immune response plays a critical role in defense against E. coli K1 infection; however, the mechanism underlying this recruitment remains unclear. In this study, we report that microglia and astrocytes are activated in response to stimulation by E. coli K1 and/or E. coli K1-derived outer membrane vesicles (OMVs) and work collaboratively to drive neutrophil recruitment to the CNS. Microglial activation results in the release of the pro-inflammatory cytokine TNF-α, which activates astrocytes, resulting in the production of CXCL1, a chemokine critical for recruiting neutrophils. Mice lacking either microglia or TNF-α exhibit impaired production of CXCL1, impaired neutrophil recruitment, and an increased CNS bacterial burden. C-X-C chemokine receptor 2 (CXCR2)-expressing neutrophils primarily respond to CXCL1 released by astrocytes. This study provides further insights into how immune responses drive neutrophil recruitment to the brain to combat E. coli K1 infection. In addition, we show that direct recognition of E. coli K1 by microglia is prevented by the K1 capsule. This study also reveals that OMVs are sufficient to induce microglial activation.

IL-21/IL-21R Regulates the Neutrophil-Mediated Pathologic Immune Response during Chlamydial Respiratory Infection

IL-21/IL-21R was documented to participate in the regulation of multiple infection and inflammation. During Chlamydia muridarum (C. muridarum) respiratory infection, our previous study had revealed that the absence of this signal induced enhanced resistance to infection with higher protective Th1/Th17 immune responses. Here, we use the murine model of C. muridarum respiratory infection and IL-21R deficient mice to further identify a novel role of IL-21/IL-21R in neutrophilic inflammation. Resistant IL-21R−/− mice showed impaired neutrophil recruitment to the site of infection. In the absence of IL-21/IL-21R, pulmonary neutrophils also exhibited reduced activation status, including lower CD64 expression, MPO activity, and neutrophil-produced protein production. These results correlated well with the decrease of neutrophil-related chemokines (KC and MIP-2), inflammatory cytokines (IL-6, IL-1β, and TNF-α), and TLR/MyD88 pathway mediators (TLR2, TLR4, and MyD88) in infected lungs of IL-21R−/− mice than normal mice. Complementarily, decreased pulmonary neutrophil infiltration, activity, and levels of neutrophilic chemotactic factors and TLR/MyD88 signal in infected lungs can be corrected by rIL-21 administration. These results revealed that IL-21/IL-21R may aggravate the neutrophil inflammation through regulating TLR/MyD88 signal pathway during chlamydial respiratory infection.

Outer membrane vesicles as carriers for bacterial extracellular ATP modulate local and remote inflammation in peritoneal sepsis

Abstract Objective Adenosine triphosphate (ATP) may be released to the extracellular space and activate specific purinergic receptors to modulate inflammatory responses. In addition to eukaryotes, ATP is also released by bacteria. This bacterial extracellular ATP (eATP) subsequently may act as an inter-kingdom signaling molecule between bacteria and its host. How ATP is secreted by bacteria and its consequences in systemic infection such as sepsis remains unclear. This study aims to determine how ATP is secreted by bacteria and to elucidate its interaction with the host immune system in a model of peritoneal sepsis. Methods Bacteria from septic patients were isolated from peritoneal washes. Peritoneal sepsis in mice was induced by cecal ligation and puncture. Peritoneal fluid was collected, (an)aerobically plated and colonies were identified by 16S-sequencing. EATP was periodically measured from bacteria culture supernatant using a luciferin-luciferase assay. Bacteria in culture were counted by plating and the amount of live/dead bacteria was assessed using flow cytometry. To specifically assess the role of bacterial eATP in vivo, E. coli K-12 BW25113 parental strain from the keio collection was used, transformed with a plasmid coding for an apyrase and i.p. injected. Furthermore, a hypervesiculation mutant of this keio collection was used to collect outer membrane vesicles (OMV). These OMV were then loaded with ATP using electroporation and injected i.p in mice. Results All bacteria that were isolated from human or mouse peritoneal washes were able to secrete ATP in a growth-phase dependent manner in vitro. In addition to passive release by dying bacteria, we have identified that the amount of eATP is connected with the formation of outer membrane vesicles (OMV) as shown by the E. coliΔtolB hypervesiculation mutant. Whole bacterial eATP led to a significant worse outcome in mice suffering from peritoneal sepsis. In addition, bacterial eATP specifically delivered via OMV worsened septic outcome as well, critically modulating neutrophil recruitment. At the infection site, ATP-loaded OMV led to an impaired recruitment of neutrophils whereas in the lung they led to an elevated infiltration. Conclusion OMV serve as a carrier of bacterial wall LPS on its surface as well as bacterial eATP as its content and specifically alter the outcome during sepsis influencing neutrophils locally as well as at remote sites.

Neutrophil dynamics in the tumor microenvironment determines therapy efficacy and is regulated by microbiota

Abstract Neutrophils play an important role in cancer progression and both pro- and antitumorigenic functions have been described. However, their role in response to therapy and whether environmental signals modulate their function in the tumor microenvironment (TME) remain unclear. Here, we show that neutrophil content in the TME defines the response to chemo- and immunotherapy. Neutrophil depletion or recruitment blockade impaired the response, while overexpression of neutrophil-attracting chemokines rendered non-responder tumors susceptible to therapy. Importantly, we demonstrate that tumor neutrophils are a heterogeneous and dynamic population that differ from those found in blood or bone marrow, and we identified a neutrophil subset that correlates with therapy response. Additionally, we found that mice lacking microbiota have impaired neutrophil recruitment and function associated with reduced therapy efficacy. Mechanistically, administration of a microbiota-derived NOD2 ligand to mice lacking microbiota was sufficient to restore neutrophil dynamics and response to therapy. We confirmed tumor neutrophil heterogeneity in cancer patients and showed that while total neutrophil content had no predictive value for patient’s overall survival, two specific neutrophil subsets associated with better outcome for pancreatic cancer patients. Remarkably, the subset enriched in non-responder germ-free tumors in our preclinical studies associated with a similar poor outcome in colorectal cancer patients. Collectively, our findings highlight the importance of characterizing neutrophil heterogeneity in the TME and the contribution of microbiota in shaping these cells to predict a successful therapy outcome. Supported by NIH Intramural Program

The role of IL-1a during early-stage Pseudomonas aeruginosa infection of the corneas

Abstract Pseudomonas aeruginosa is an important cause of dermal, pulmonary and ocular disease. Our studies have focused on P. aeruginosa infections of the cornea as a major cause of blinding microbial infections. The infection leads to an influx of innate immune cells with neutrophils making up to 90% of recruited cells during early stages. We previously reported that the proinflammatory cytokines IL-1a and IL-1b were elevated during infection. Infected Il1b−/− mice developed more severe corneal disease that is associated with impaired bacterial killing as a result of defective neutrophil recruitment to the corneas. We also reported that neutrophils are an important source of IL-1b and IL-1a. To examine the role of IL-1a in Pseudomonas aeruginosa keratitis, we inoculated corneas of C57BL/6 (WT), Il1a−/−, Il1b−/− and Il1a−/−Il1b−/− (DKO) mice with 5×10^4 ExoS expressing P. aeruginosa (PAO1 strain). We identified neutrophils and monocytes as the main producers of IL-1a which peaked at 24 hours post infection (hpi). Depletion of neutrophils or monocytes led to more severe corneal disease and higher bacterial burden. Il1b−/− and DKO mice have significantly higher bacterial burden that was consistent with impaired neutrophil and monocyte recruitment to the corneas. Surprisingly, Il1a−/− mice had the opposite phenotype with significantly less bacteria than WT mice. There were no significant differences in neutrophil recruitment. However, we hypothesize that Il1a−/− neutrophils have enhanced antimicrobial functions that resulted in more effective bacterial killing which we are addressed by RNA sequencing of neutrophils isolated from WT or Il1a−/− corneas. Supported by grants (EP) RO1: EY18612 and (BR) F31: EY032312-01

Soluble uric acid inhibits β2 integrin–mediated neutrophil recruitment in innate immunity

AbstractNeutrophils are key players during host defense and sterile inflammation. Neutrophil dysfunction is a characteristic feature of the acquired immunodeficiency during kidney disease. We speculated that the impaired renal clearance of the intrinsic purine metabolite soluble uric acid (sUA) may account for neutrophil dysfunction. Indeed, hyperuricemia (HU, serum UA of 9-12 mg/dL) related or unrelated to kidney dysfunction significantly diminished neutrophil adhesion and extravasation in mice with crystal- and coronavirus-related sterile inflammation using intravital microscopy and an air pouch model. This impaired neutrophil recruitment was partially reversible by depleting UA with rasburicase. We validated these findings in vitro using either neutrophils or serum from patients with kidney dysfunction–related HU with or without UA depletion, which partially normalized the defective migration of neutrophils. Mechanistically, sUA impaired β2 integrin activity and internalization/recycling by regulating intracellular pH and cytoskeletal dynamics, physiological processes that are known to alter the migratory and phagocytic capability of neutrophils. This effect was fully reversible by blocking intracellular uptake of sUA via urate transporters. In contrast, sUA had no effect on neutrophil extracellular trap formation in neutrophils from healthy subjects or patients with kidney dysfunction. Our results identify an unexpected immunoregulatory role of the intrinsic purine metabolite sUA, which contrasts the well-known immunostimulatory effects of crystalline UA. Specifically targeting UA may help to overcome certain forms of immunodeficiency, for example in kidney dysfunction, but may enhance sterile forms of inflammation.

Priming of microvascular endothelium by microbiota-derived metabolites regulates neutrophil trafficking to the liver during sepsis

Abstract During systemic bacterial infections and sepsis, neutrophils are recruited en mass into the microcirculation of the liver and lungs as part of a coordinated intravascular response to defend the bloodstream against bacterial pathogens. The mechanisms that program this response have not been elucidated. The gut microbiota regulates immune cell development and function. In extra-intestinal organs, the microbiota conducts long-distance communication with immune cells through the release of metabolites into the bloodstream. While gut microbes augment granulopoiesis in the bone marrow, the role the microbiota and commensal-derived metabolites on neutrophil trafficking is poorly understood. Using confocal intravital microscopy, we observed that germ-free (GF) mice had impaired neutrophil recruitment to the liver, but not the lungs, during sepsis. Analysis of microbial metabolites in the gut, portal circulation (between gut and liver), and systemic circulation revealed that commensal-derived D-lactate levels are high in the gut and portal circulation, but nearly absent in the systemic circulation. Reconstitution of gut-to-liver D-lactate signalling by enteral administration restored neutrophil trafficking to the liver. In contrast, D-lactate did not impact neutrophil infiltration to the lungs, systemic levels of pro-inflammatory mediators or granulopoiesis. Instead, commensal D-lactate primed liver endothelium to express adhesion molecules required for neutrophil adhesion and crawling. Taken together, we show that compartmentalized molecular communication between gut microbes and the liver primes the microvascular endothelium for neutrophil recruitment in response to systemic bacterial infection and sepsis.

198 TNF directs protective neutrophil and IL-17+ γδ T cell responses against Staphylococcus aureus skin infections

Staphylococcus aureus is the leading cause of skin and soft tissue infections and has become a major health burden due to the emergence of antibiotic-resistant strains. To develop alternative therapies to antibiotics, we sought to understand the protective immune mechanisms against S. aureus skin infections mediated by tumor necrosis factor (TNF). TNF is a proinflammatory cytokine that is rapidly induced upon S. aureus exposure and whose inhibition is associated with increased risk of S. aureus infections in humans. However, the contribution of TNF or the cognate receptors, TNFR1 and TNFR2, to host defense against S. aureus skin infections is unclear. Therefore, to determine the host defense role of TNF, we used an in vivo mouse model of S. aureus skin infection whereby TNF, TNFR1, or TNFR2 deficient mice and wildtype (wt) mice were intradermally injected with bioluminescent S. aureus and monitored for 14 days. TNF, TNFR1, and TNFR2 deficient mice exhibited increased lesion sizes and bacterial burden compared to wt mice, suggesting TNF is important against S. aureus skin infections and TNFR1 and TNFR2 have non-redundant roles in TNF-mediated immunity. Since TNF has been reported to contribute to neutrophil trafficking and IL-17 expression, we hypothesized that TNF promotes the previously identified protective neutrophil abscess formation and IL-17+ γδ T cell responses against S. aureus skin infection. In fact, TNF deficient mice had significantly impaired neutrophil recruitment, abscess formation, and diminished skin-infiltrating IL-17+ γδ T cells compared to wt mice. Taken together, these findings indicated that differential TNF signaling by TNFR1 and TNFR2 directs protective neutrophil and IL-17+ γδ T cell responses during S. aureus skin infections, which has implications in the development of novel immune-based therapies as alternatives to antibiotic treatment against S. aureus and potentially other bacterial infections.

Interactions Between Neutrophils and Periodontal Pathogens in Late-Onset Periodontitis.

Late-onset periodontitis is associated with a series of inflammatory reactions induced by periodontal pathogens, such as Porphyromonas gingivalis, a keystone pathogen involved in periodontitis. Neutrophils are the most abundant leukocytes in the periodontal pocket/gingival crevice and inflamed periodontal tissues. They form a “wall” between the dental plaque and the junctional epithelium, preventing microbial invasion. The balance between neutrophils and the microbial community is essential to periodontal homeostasis. Excessive activation of neutrophils in response to periodontal pathogens can induce tissue damage and lead to periodontitis persistence. Therefore, illuminating the interactions between neutrophils and periodontal pathogens is critical for progress in the field of periodontitis. The present review aimed to summarize the interactions between neutrophils and periodontal pathogens in late-onset periodontitis, including neutrophil recruitment, neutrophil mechanisms to clear the pathogens, and pathogen strategies to evade neutrophil-mediated elimination of bacteria. The recruitment is a multi-step process, including tethering and rolling, adhesion, crawling, and transmigration. Neutrophils clear the pathogens mainly by phagocytosis, respiratory burst responses, degranulation, and neutrophil extracellular trap (NET) formation. The mechanisms that pathogens activate to evade neutrophil-mediated killing include impairing neutrophil recruitment, preventing phagocytosis, uncoupling killing from inflammation, and resistance to ROS, degranulation products, and NETs.

Increased Abundance of Tumour-Associated Neutrophils in HPV-Negative Compared to HPV-Positive Oropharyngeal Squamous Cell Carcinoma Is Mediated by IL-1R Signalling.

The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome.

DIETARY FIBER DEPRIVATION QUELLS COLONIC INFLAMMATION BY TARGETING GUT PATHOBIONTS IN A NEW MODEL OF CROHN’S DISEASE

Abstract Crohn’s disease (CD) is a chronic intestinal inflammatory disorder that results from a dysregulated mucosal immune response to symbiotic bacteria in genetically susceptible individuals. While genetic predispositions to CD have been well documented, the overall contribution of genetic factors to disease development remains unclear. Indeed, abnormal interactions between microbes and the immune system have been identified as the core defect leading to chronic intestinal inflammation. We used a “multi-hit” genetic approach to study the complex interplay between host immunity and microbes in the context of CD. We have found that the combined deficiency of NOD2 and phagocyte NADPH oxidase (i.e. CYBB), two CD susceptibility genes, triggers microbiota-dependent and spontaneous TH1-type colitis in mice that is accompanied by the pathological hallmarks seen in CD patients. Disease was induced by Mucispirillum schaedleri, a gram-negative mucus-dwelling anaerobe. The absence of the two anti-microbial genes (i.e. NOD2 and CYBB) resulted in a marked accumulation of M. schaedleri in the gut lumen, the colonic mucus layer and ultimately a focal penetration of the intestinal wall. Accumulation of M. schaedleri was associated with impaired recruitment of neutrophils in the intestine and subsequently inefficient elimination of the bacterium. Among the environmental factors associated with CD, diet shapes the composition of the gut microbiota and can be used to manage disease symptoms. Our experiments have shown that consumption of a fiber-free diet leads to a depletion of the colonic mucus layer and increased proximity between microbes and the gut epithelium. Interestingly, these experimental conditions are effective at inhibiting intestinal inflammation observed in our model of CD-like colitis when used both as a preventive and as a therapeutic approach. In addition to shaping the overall gut microbiota composition, this dietary intervention specifically alters the abundance and/or localization of the disease-causing M. schaedleri in the double mutant mice. Our results provide direct evidence that a specific intestinal microbe can, in conjunction with impaired bacterial clearance, trigger CD-like disease. Our results also indicate that dietary approaches can be successfully used to target microbial-driven intestinal pathology.

Distinct Tissue Damage and Microbial Cues Drive Neutrophil and Macrophage Recruitment to Thermal Injury.

SummaryTissue damage triggers a rapid innate immune response that mediates host defense. Previously we reported that thermal damage of the larval zebrafish fin disrupts collagen organization and induces a robust and potentially damaging innate immune response. The mechanisms that drive damaging versus protective neutrophil inflammation in interstitial tissues remain unclear. Here we identify distinct cues in the tissue microenvironment that differentially drive neutrophil and macrophage responses to sterile injury. Using live imaging, we found a motile zone for neutrophils, but not macrophages, in collagen-free regions and identified a specific role for interleukin-6 (IL-6) receptor signaling in neutrophil responses to thermal damage. IL-6 receptor mutants show impaired neutrophil recruitment to sterile thermal injury that was not present in tissues infected with Pseudomonas aeruginosa. These findings identify distinct signaling networks during neutrophil recruitment to sterile and microbial damage cues and provide a framework to limit potentially damaging neutrophil inflammation.

An IL-17F.S65L Knock-In Mouse Reveals Similarities and Differences in IL-17F Function in Oral Candidiasis: A New Tool to Understand IL-17F.

Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus Candida albicans IL-17R signaling is essential to prevent OPC in mice and humans, but the individual roles of its ligands, IL-17A, IL-17F, and IL-17AF, are less clear. A homozygous IL-17F deficiency in mice does not cause OPC susceptibility, whereas mice lacking IL-17A are moderately susceptible. In humans, a rare heterozygous mutation in IL-17F (IL-17F.S65L) was identified that causes chronic mucocutaneous candidiasis, suggesting the existence of essential antifungal pathways mediated by IL-17F and/or IL-17AF. To investigate the role of IL-17F and IL-17AF in more detail, we exploited this "experiment of nature" by creating a mouse line bearing the homologous mutation in IL-17F (Ser65Leu) by CRISPR/Cas9. Unlike Il17f-/- mice that are resistant to OPC, Il17fS65L/S65L mice showed increased oral fungal burdens similar to Il17a -/- mice. In contrast to humans, however, disease was only evident in homozygous, not heterozygous, mutant mice. The mutation was linked to modestly impaired CXC chemokine expression and neutrophil recruitment to the infected tongue but not to alterations in oral antimicrobial peptide expression. These findings suggest mechanisms by which the enigmatic cytokine IL-17F contributes to host defense against fungi. Moreover, because these mice do not phenocopy Il17f-/- mice, they may provide a valuable tool to interrogate IL-17F and IL-17AF function in vivo in other settings.