Neutrophil Chemotactic Factor Research Articles

LECT2 Deletion Exacerbates Liver Steatosis and Macrophage Infiltration in a Male Mouse Model of LPS-mediated NASH.

Leukocyte cell-derived chemotaxin 2 (LECT2) is a protein initially isolated as a neutrophil chemotactic factor. We previously found that LECT2 is an obesity-associated hepatokine that senses liver fat and induces skeletal muscle insulin resistance. In addition, hepatocyte-derived LECT2 activates macrophage proinflammatory activity by reinforcing the lipopolysaccharide (LPS)-induced c-Jun N-terminal kinase signaling. Based on these findings, we examined the effect of LECT2 deletion on nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) caused by bacterial translocation. We created the bacterial translocation-mediated NAFLD/NASH model using LECT2 knockout mice (LECT2 KO) with 28 times a low-dose LPS injection under high-fat diet feeding conditions. LECT2 deletion exacerbated steatosis and significantly reduced p38 phosphorylation in the liver. In addition, LECT2 deletion increased macrophage infiltration with decreased M1/M2 ratios. LECT2 might contribute to protecting against lipid accumulation and macrophage activation in the liver under pathological conditions, which might be accomplished via p38 phosphorylation. This study provides novel aspects of LECT2 in the bacterial translocation-mediated NAFLD/NASH model.

Porcine cis-acting lnc-CAST positively regulates CXCL8 expression through histone H3K27ac

The chemokine CXCL8, also known as the neutrophil chemotactic factor, plays a crucial role in mediating inflammatory responses and managing cellular immune reactions during viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) primarily infects pulmonary alveolar macrophages (PAMs), leading to acute pulmonary infections. In this study, we explored a novel long non-coding RNA (lncRNA), termed lnc-CAST, situated within the Cxcl8 gene locus. This lncRNA was found to be highly expressed in porcine macrophages. We observed that both lnc-CAST and CXCL8 were significantly upregulated in PAMs following PRRSV infection, and after treatments with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Furthermore, we noticed a concurrent upregulation of lnc-CAST and CXCL8 expression in lungs of PRRSV-infected pigs. We then determined that lnc-CAST positively influenced CXCL8 expression in PAMs. Overexpression of lnc-CAST led to an increase in CXCL8 production, which in turn enhanced the migration of epithelial cells and the recruitment of neutrophils. Conversely, inhibiting lnc-CAST expression resulted in reduced CXCL8 production in PAMs, leading to decreased migration levels of epithelial cells and neutrophils. From a mechanistic perspective, we found that lnc-CAST, localized in the nucleus, facilitated the enrichment of histone H3K27ac in CXCL8 promoter region, thereby stimulating CXCL8 transcription in a cis-regulatory manner. In conclusion, our study underscores the pivotal critical role of lnc-CAST in regulating CXCL8 production, offering valuable insights into chemokine regulation and lung damage during PRRSV infection.

CXCL12+ dermal fibroblasts promote neutrophil recruitment and host defense by recognition of IL-17.

The skin provides an essential barrier for host defense through rapid action of multiple resident and recruited cell types, but the complex communication network governing these processes is incompletely understood. To define these cell-cell interactions more clearly, we performed an unbiased network analysis of mouse skin during invasive S. aureus infection and revealed a dominant role for CXCL12+ fibroblast subsets in neutrophil communication. These subsets predominantly reside in the reticular dermis, express adipocyte lineage markers, detect IL-17 and TNFα, and promote robust neutrophil recruitment through NFKBIZ-dependent release of CXCR2 ligands and CXCL12. Targeted deletion of Il17ra in mouse fibroblasts resulted in greatly reduced neutrophil recruitment and increased infection by S. aureus. Analogous human CXCL12+ fibroblast subsets abundantly express neutrophil chemotactic factors in psoriatic skin that are subsequently decreased upon therapeutic targeting of IL-17. These findings show that CXCL12+ dermal immune acting fibroblast subsets play a critical role in cutaneous neutrophil recruitment and host defense.

Neutrophils are indispensable for adverse cardiac remodeling in heart failure

Persistent immune activation contributes significantly to left ventricular (LV) dysfunction and adverse remodeling in heart failure (HF). In contrast to their well-known essential role in acute myocardial infarction (MI) as first responders that clear dead cells and facilitate subsequent reparative macrophage polarization, the role of neutrophils in the pathobiology of chronic ischemic HF is poorly defined. To determine the importance of neutrophils in the progression of ischemic cardiomyopathy, we measured their production, levels, and activation in a mouse model of chronic HF 8 weeks after permanent coronary artery ligation and large MI. In HF mice, neutrophils were more abundant both locally in failing myocardium (more in the border zone) and systemically in the blood, spleen, and bone marrow, together with increased BM granulopoiesis. There were heightened stimuli for neutrophil recruitment and trafficking in HF, with increased myocardial expression of the neutrophil chemoattract chemokines CXCL1 and CXCL5, and increased neutrophil chemotactic factors in the circulation. HF neutrophil NETotic activity was increased in vitro with coordinate increases in circulating neutrophil extracellular traps (NETs) in vivo. Neutrophil depletion with either antibody-based or genetic approaches abrogated the progression of LV remodeling and fibrosis at both intermediate and late stages of HF. Moreover, analogous to murine HF, the plasma milieu in human acute decompensated HF strongly promoted neutrophil trafficking. Collectively, these results support a key tissue-injurious role for neutrophils and their associated cytotoxic products in ischemic cardiomyopathy and suggest that neutrophils are potential targets for therapeutic immunomodulation in this disease.

Inhibition of neutrophil elastase prevents cigarette smoke exposure-induced formation of neutrophil extracellular traps and improves lung function in a mouse model of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is an important public health challenge worldwide, and is usually caused by significant exposure to noxious agents, particularly cigarette smoke. Recent studies have revealed that excessive production of neutrophil extracellular traps (NETs) in the airways is associated with disease severity in COPD patients. NETs are extracellular neutrophil-derived structures composed of chromatin fibers decorated with histones and granule proteases including neutrophil elastase (NE). However, the effective prevention of NET formation in COPD remains elusive. Here, we demonstrated that treatment with GW311616A, a potent and selective inhibitor of NE, prevented cigarette smoke extract (CSE)-induced NET formation in human neutrophils by blocking NE nuclear translocation and subsequent chromatin decondensation. Inhibition of NE also abrogated CSE-induced ROS production and migration impairment of neutrophils. Administration of GW311616A in vivo substantially reduced pulmonary generation of NETs while attenuating the key pathological changes in COPD, including airway leukocyte infiltration, mucus-secreting goblet cell hyperplasia, and emphysema-like alveolar destruction in a mouse model of COPD induced by chronic cigarette smoke exposure. Mice treated with GW311616A also showed significant attenuation of neutrophil numbers and percentages and the levels of neutrophil chemotactic factors (LTB4, KC, and CXCL5) and proinflammatory cytokines (IL-1β, and TNF-α) in bronchoalveolar lavage fluid compared to mice treated with cigarette smoke exposure only. Furthermore, GW311616A treatment considerably improved lung function in the COPD mouse model, including preventing the decline of FEV100/FVC and delta PEF as well as inhibiting the increase in FRC, TLC, and FRC/TLC. Overall, our study suggests that NE plays a critical role in cigarette smoke-induced NET formation by neutrophils and that inhibition of NE is a promising strategy to suppress NET-mediated pathophysiological changes in COPD.

125P Serum CXCL8 forms a pro-tumor phenotype of circulating neutrophils in clear cell kidney cancer

CXCL8 is a neutrophil chemotactic factor (Nph), which is a key effector cell of innate immunity and plays a crucial role in various inflammatory diseases. CXCL8 regulates pathological angiogenesis, tumor growth and metastasis and enhances the protumor effect of Nph (Gonzalez-Aparicio M., 2020). At the same time, protumor Nph (N1) are characterized by reduced expression of chemokines (Sagiv J.Y., 2015). The study was aimed at assessing the expression of the CXCL8 gene by circulating Nph and the serum level of CXCL8 at different stages of clear cell kidney cancer.

The Importance of CXCL1 in the Physiological State and in Noncancer Diseases of the Oral Cavity and Abdominal Organs.

CXCL1 is a CXC chemokine, CXCR2 ligand and chemotactic factor for neutrophils. In this paper, we present a review of the role of the chemokine CXCL1 in physiology and in selected major non-cancer diseases of the oral cavity and abdominal organs (gingiva, salivary glands, stomach, liver, pancreas, intestines, and kidneys). We focus on the importance of CXCL1 on implantation and placentation as well as on human pluripotent stem cells. We also show the significance of CXCL1 in selected diseases of the abdominal organs, including the gastrointestinal tract and oral cavity (periodontal diseases, periodontitis, Sjögren syndrome, Helicobacter pylori infection, diabetes, liver cirrhosis, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), HBV and HCV infection, liver ischemia and reperfusion injury, inflammatory bowel disease (Crohn’s disease and ulcerative colitis), obesity and overweight, kidney transplantation and ischemic-reperfusion injury, endometriosis and adenomyosis).

LECT2: A pleiotropic and promising hepatokine, from bench to bedside.

LECT2 (leucocyte cell‐derived chemotaxin 2) is a 16‐kDa protein mainly produced by hepatocytes. It was first isolated in PHA‐activated human T‐cell leukaemia SKW‐3 cells and originally identified as a novel neutrophil chemotactic factor. However, many lines of studies suggested that LECT2 was a pleiotropic protein, it not only functioned as a cytokine to exhibit chemotactic property, but also played multifunctional roles in some physiological conditions and pathological abnormalities, involving liver regeneration, neuronal development, HSC(haematopoietic stem cells) homeostasis, liver injury, liver fibrosis, hepatocellular carcinoma, metabolic disorders, inflammatory arthritides, systemic sepsis and systemic amyloidosis. Among the above studies, it was discovered that LECT2 could be a promising molecular biomarker and therapeutic target. This review summarizes LECT2‐related receptors and pathways, basic and clinical researches, primarily in mice and human, for a better comprehension and management of these diseases in the future.

Interleukin-34 permits Porphyromonas gingivalis survival and NF-κB p65 inhibition in macrophages.

Interleukin-34 (IL-34) is a cytokine that supports the viability and differentiation of macrophages. An important cytokine for the development of epidermal immunity, IL-34, is present and plays a role in the immunity of the oral environment. IL-34 has been linked to inflammatory periodontal diseases, which involve innate phagocytes, including macrophages. Whether IL-34 can alter the ability of macrophages to effectively interact with oral microbes is currently unclear. Using macrophages derived from human blood monocytes with either the canonical cytokine colony-stimulating factor (CSF)1 or IL-34, we compared the ability of the macrophages to phagocytose, kill, and respond through the production of cytokines to the periodontal keystone pathogen Porphyromonas gingivalis. While macrophages derived from both cytokines were able to engulf the bacterium equally, IL-34-derived macrophages were much less capable of killing internalized P.gingivalis. Of the macrophage cell surface receptors known to interact with P.gingivalis, dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin was found to have the largest variation between IL-34- and CSF1-derived macrophages. We also found that upon interaction with P.gingivalis, IL-34-derived macrophages produced significantly less of the neutrophil chemotactic factor IL-8 than macrophages derived in the presence of CSF1. Mechanistically, we identified that the levels of IL-8 corresponded with P. gingivalis survival and dephosphorylation of the major transcription factor NF-κB p65. Overall, we found that macrophages differentiated in the presence of IL-34, a dominant cytokine in the oral gingiva, have a reduced ability to kill the keystone pathogen P.gingivalis and may be susceptible to specific bacteria-mediated cytokine modification.

Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1β and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

Cytokines in Febrile Diseases.

The human body has a perfect thermoregulatory system to meet the needs of normal life activities. The central regulation of body temperature is mainly explained by the theory of "setting point (setpoint, SP)". Fever is a positive but nonspecific response of the body to infections and other pyrogens, which causes immune cells to release cytokines, leading to a brain protein-mediated rise in body temperature. Cytokines can be roughly divided into 2 categories: proinflammatory cytokines and anti-inflammatory cytokines. IL-1, TNF-α, and IL-6 are proinflammatory cytokines, whereas IL-4 and IL-10 are anti-inflammatory cytokines. IL-2 is a cytokine that can both activate and inhibit immunity. IL-8 is a neutrophil chemotactic factor, and IFN is a cytokine that plays a key role in the proper induction and maintenance of innate and acquired immunity. This article reviews the pathophysiological characteristics of fever and the cytokines related to fever (IL-2, 4, 6, 8, 10, IFN, TNF, etc.).

Production of Hematopoietic Stem Cell-Chemotactic Factor by Bone Marrow Stromal Cells

Chemotactic factors produced by stromal cells in the bone marrow are characterized. Two kinds of factors produced by stromal cell lines are identified using blind-well Boyden's Chambers; one is a neutrophil-chemotactic factor and the other a hematopoietic stem cell (HSC)-chemotactic factor. The latter attracts blastic cells in a low-density fraction, which are Thy1lo, wheat germ agglutinin (WGA)hi, H-2Khi, Ly-1-, Ly-2-, L3T4-, Ly5-, and slg-. The molecular weight of this HSC-chemotactic factor is estimated to be more than 200 kD. Putative cytokines and growth factors, such as granulocyte colony-stimulating factor (CSF), macrophage CSF, granulocyte-macrophage CSF, stem cell factor (SCF), interleukin (IL)-6, and IL-3, do not possess HSC-chemotactic activity. These findings strongly suggest that bone marrow stromal cells produce a new factor that attracts HSCs.

Alpha 1 Antitrypsin-Deficient Macrophages Have Impaired Efferocytosis of Apoptotic Neutrophils.

Alpha 1 antitrypsin deficiency (AATD) is an autosomal co-dominant disorder characterized by a low level of circulating AAT, which significantly reduces protection for the lower airways against proteolytic burden caused by neutrophils. Neutrophils, which are terminally differentiated innate immune cells and play a critical role to clear pathogens, accumulate excessively in the lung of AATD individuals. The neutrophil burden in AATD individuals increases the risk for early-onset destructive lung diseases by producing neutrophil products such as reactive oxygen radicals and various proteases. The level of AAT in AATD individuals is not sufficient to inhibit the activity of neutrophil chemotactic factors such as CXCL-8 and LTB4, which could lead to alveolar neutrophil accumulation in AATD individuals. However, as neutrophils have a short lifespan, and apoptotic neutrophils are rapidly cleared by alveolar macrophages that outnumber the apoptotic neutrophils in the pulmonary alveolus, the increased chemotaxis activity does not fully explain the persistent neutrophil accumulation and the resulting chronic inflammation in AATD individuals. Here, we propose that the ability of alveolar macrophages to clear apoptotic neutrophils is impaired in AATD individuals and it could be the main driver to cause neutrophil accumulation in their lung. This study demonstrates that Z-AAT variant significantly increases the expression of pro-inflammatory cytokines including CXCL-8, CXCL1, LTB4, and TNFα in LPS-treated macrophages. These cytokines play a central role in neutrophil recruitment to the lung and in clearance of apoptotic neutrophils by macrophages. Our result shows that LPS treatment significantly reduces the efferocytosis ability of macrophages with the Z-AAT allele by inducing TNFα expression. We incubated monocyte-derived macrophages (MDMs) with apoptotic neutrophils and found that after 3 h of co-incubation, the expression level of CXCL-8 is reduced in M-MDMs but increased in Z-MDMs. This result shows that the expression of inflammatory cytokines could be increased by impaired efferocytosis. It indicates that the efferocytosis ability of macrophages plays an important role in regulating cytokine expression and resolving inflammation. Findings from this study would help us better understand the multifaceted effect of AAT on regulating neutrophil balance in the lung and the underlying mechanisms.

Evidence of Microangiopathy in Children with Sars-Cov-2 Regardless of Clinical Presentation

Introduction: During the Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), 3 distinct phenotypes have emerged in children. The majority of children have mild or no symptoms. Similar to adults, a minority of children can be severely affected with respiratory distress requiring intensive care. Finally, they may develop a phenomenon presumed unique to children termed Multisystem Inflammatory Syndrome in Children (MIS-C). MIS-C is a hyperinflammatory syndrome characterized by fever and organ dysfunction (particularly cardiac) in the setting of recent COVID-19 infection. Reports from the adult literature have invoked thrombotic microangiopathy (TMA) and complement activation as a potential cause for severe manifestations of COVID-19 (Zhang et al. NEJM. 2020; Campbell et al. Circulation 2020). Soluble C5b9 (sC5b-9), the terminal complement complex, has been implicated as a marker of hematopoietic stem cell transplant associated TMA (HSCT-TMA; Jodele et al. Blood 2014). We sought to elucidate the role of terminal complement activation and TMA in the different pediatric disease phenotypes.Methods: We enrolled children admitted to the Children's Hospital of Philadelphia during the COVID-19 pandemic who had evidence of SARS-CoV-2 infection on reverse transcriptase polymerase chain reaction (RT-PCR) from mucosa, or met clinical criteria for MIS-C. Patients (pts) were classified in to 3 categories: minimal COVID-19 symptoms or incidental finding of SARS-CoV-2 infection, severe COVID-19 requiring ventilatory support, or MIS-C. To investigate the role of TMA in children with COVID-19 we measured sC5b-9 in plasma of pts with the 3 manifestations of SARS-CoV-2, and in healthy controls. sC5b9 was measured in triplicate at two dilutions by ELISA. Proinflammatory cytokines were measured using V-Plex Pro-inflammatory Panel 1 Human Kits and analyzed on a QuickPlex SQ120. P-values were computed using Dunn's multiple comparisons test after Kruskal-Wallis testing. Blood smears were examined by a hematologist and hematopathologist for schistocytes.Results: 50 pts were enrolled on whom complete sC5b9 data were available: minimal COVID-19 (N=18), severe COVID-19 (N=11), and MIS-C (N=21). Plasma was obtained on healthy controls (N=26). The median sC5b9 level in healthy controls (57 ng/mL) differed significantly (p<0.001 in each case; Figure 1A) from that in pts with minimal disease (392 ng/mL), severe disease (646 ng/mL), and MIS-C (630 ng/mL); differences between MIS-C, minimal, and severe were not statistically significant. Elevations in sC5b9 correlated in a statistically significant manner with the maximum creatinine and blood urea nitrogen (BUN) measured during hospitalization (Figure 1B&C), but not age (p=0.512). sC5b9 did not correlate with lactate dehydrogenase (LDH), nor with the lowest levels of fibrinogen, hemoglobin or platelet counts. Of pts with available data, 19/26 (73.1%) had elevated LDH, 2/31 (6.4%) had hypofibrinogenemia, 35/47 (74.5%) were anemic, and 28/47 (59.6%) were thrombocytopenic.Pro-inflammatory cytokines were measured. Of particular interest to TMA is the neutrophil chemotactic factor IL-8, because of its role as a marker of endothelial damage (Dvorak et al. Front Pediatr 2019). Levels of IL-8 differed significantly between pts with MIS-C (p=0.0166) or pts with severe COVID-19 (p=0.0079), when compared to minimal COVID-19 pts; but not between pts with MIS-C and severe disease (p = 0.99).Blood smears were available on 34 patients. Schistocytes were present in 13/15 (87%) patients with MIS-C, 7/8 (87%) patients with severe COVID-19 and 5/11 (45%) patients with minimal COVID-19 (χ2=6.59, p=0.037).Conclusions: We demonstrate derangements of the final common pathway of complement activation in children with the 3 presentations of SARS-CoV-2. Strikingly, sC5b9s were abnormal even in children with minimal disease or incidental infection. Renal dysfunction correlated with elevations in sC5b9, strengthening the evidence that TMA plays a role in the pathophysiology of SARS-CoV-2 infection. Future work is aimed at further characterizing the role of the complement cascade in the pathogenesis of MIS-C and COVID-19 in children. The long-term complications of endothelial damage and complement activation are unknown and extended follow-up is warranted.Figure 1DisclosuresDiorio:Children's Hospital of Philadelphia: Research Funding; University of Pennsylvania: Research Funding. Lambert:22qSociety: Consultancy; RDMD ITP study: Consultancy; Octapharma: Consultancy, Research Funding; Educational Concepts in Medicine: Consultancy; Shionogi: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Argenix: Consultancy; CdLS Foundation: Consultancy; Sysmex: Research Funding; Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; ClinGen: Honoraria; Platelet Disorder Support Association (PDSA): Consultancy; AstraZeneca: Research Funding; Bayer: Consultancy; ITP Australia: Consultancy. Henrickson:Horizon Pharma: Other: ad hoc board meeting. Odom John:Burroughs Wellcome: Research Funding; NIAID: Research Funding. Bassiri:CSL Behring: Other: Spouse receives stocks . Behrens:NIH/NIAID: Research Funding. Teachey:Janssen: Consultancy; Amgen: Consultancy; La Roche: Consultancy; Sobi: Consultancy.

Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis.

Despite the introduction of biologics, topical dithranol (anthralin) has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e. IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), antimicrobial peptides (e.g. ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), chemotactic factors for neutrophils (e.g. CXCL5, CXCL8) and neutrophilic infiltration was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs.

Identification of N-formylated peptides as the neutrophilic chemotactic factors of Mycobacterium tuberculosis

Abstract Since tuberculosis (TB) remains a major cause of human death, it is urgent to understand the pathology of TB. Lung neutrophil infiltration initiates granuloma formation and is associated with disease severity. Though several cytokines and chemokines are associated with neutrophil infiltration in chronic inflammation, neutrophil chemotactic factors of Mycobacterium tuberculosis (Mtb) remain unexplored. Thus, we performed neutrophil chemotactic assays by culturing neutrophils of healthy human blood samples and mouse bone marrow in the upper chamber of a transwell plate with 5–8 μm pores with the culture filtrates of Mtb H37Rv (CF) in the lower chamber using N-formyl-met-leu-phe (fMLF), a bacterial neutrophil chemotactic tripeptide, as a positive control. The cell numbers in the lower chamber were determined by flow cytometry as a chemotactic indicator. Our results show that CF induced chemotaxis of both human and mouse neutrophils in an Mtb culture period dependent manner. Testing CF of 11 clinical isolates of Mtb also showed neutrophil chemotactic activity. Sulfasalazine, a formylated peptide receptor inhibitor, blocked chemotaxis of neutrophils, indicating the involvement of fPRs in CF induces chemotaxis of neutrophils. Mass spectrometry analysis of CF identified three candidate N-formylated heptapeptides. Authentication of the chemotactic activities of the identified peptides with synthetic mimetics confirmed their neutrophil chemotactic activities and indicated the requirement of N-formylation for their chemotactic activities. Thus, we have identified novel formylated peptides of Mtb with neutrophil chemotactic activities, and further studies are in progress to determine their significance in TB infection.

Gene expression profiling of unresectable pancreatic cancer patients treated with gemcitabine, nab-paclitaxel, metformin, and dietary supplements (DS).

736 Background: Pancreatic cancer commonly causes weight loss and many patients take supplements to improve nutrition but also for any potential anticancer properties. We conducted a pilot trial evaluating a standardized combination of dietary supplements, metformin and gemcitabine plus nab-paclitaxel chemotherapy. Methods: The supplements were custom designed by a nutritionally trained oncologist. 17 supplements were given as 12 pills and 2 smoothie packets divided twice per day. Patients began metformin on day (D)-6 to evaluate GI tolerance before starting the DS on D-3. On D1, gemcitabine and nab-paclitaxel chemotherapy was administered at standard doses. Serum samples were collected D-6, C1D1, C3D1 and at the end of treatment. RNA was extracted from PBMC samples and a total of 48 samples were analyzed from 19 patients. NanoString Human Immune Profiling and PanCancer Pathways were performed to evaluate changes in gene expression with treatment. Results: The differential expression of genes was small with dietary supplements alone and in combination with chemotherapy. After 2 months of treatment, 17 genes were upregulated while 4 genes were down regulated. At the time of tumor resistance, 33 genes were upregulated while 17 genes were downregulated. We observed downregulation of tumor suppressor gene expression with upregulation in growth factor pathways. Interestingly, SPRY2 mRNA expression was also up regulated which functions as a negative feedback regulator of multiple receptor tyrosine kinases. Conclusions: Increasing number of genes were upregulated with continued treatment. The most common pathway affected was cell cycle and apoptosis. During the initial supplement run-in period from D-6 to C1D1, IL-8 mRNA expression was upregulated the most. IL-8 is a neutrophil chemotactic factor secreted by cells involved in the innate immune response as well as pancreatic cancer cells associated with a pro-inflammatory state. Treatment with gemcitabine and nab-paclitaxel decreased the IL-8 mRNA expression. Additional studies with longer course treatment with supplements alone would be required to explore its impact on differential gene expression.

IL8 and PMA Trigger the Regulation of Different Biological Processes in Granulocyte Activation

The molecular mechanisms driving specific regulation of neutrophils are not completely understood to date. In order to characterize fundamental granulocyte features on protein level, we analyzed changes in proteome composition as reaction to stress from cell activation processes. For this purpose, we isolated primary granulocytes from equine whole blood through density gradient centrifugation followed by sodium chloride lysis and stimulated cells for 30 min with interleukin-8 (IL8) due to its role as a chemotactic factor for neutrophils. We additionally used phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), which are primarily associated to neutrophil extracellular trap formation and release of reactive oxygen species. From mass spectrometry analysis, we identified a total of 2,032 proteins describing the whole granulocyte proteome, including 245 proteins (12% of identified proteome) newly associated to in vivo expression in primary equine granulocytes (hypothetical proteins). We also found distinct and different changes in protein abundance (ratio ≥ 2) after short stimulation of cells with various stimuli, pointing to rapid and differentiated reaction pattern. IL8 stimulation resulted in increased protein abundance of 58 proteins (3% of proteome), whereas PMA induced changed protein abundance of 207 (10 % of proteome) and LPS of 46 proteins (2% of proteome). Enrichment analyses clearly showed fundamental differences between stimuli, with primary association of IL8 stimulation to processes in immune response, receptor signaling and signal transduction. Top enrichment for PMA on the other hand pointed to vesicle mediated transport and exocytosis. Stimulation with LPS did not result in any significant enrichment. Although we detected 43% overlap of enrichment categories for IL8 and PMA stimulation, indicating that activation of neutrophils with different stimuli partly induces some similar biological processes and pathways, hierarchical clustering showed clear differences in distribution and biological relevance of clusters between the chosen stimuli. Our studies provide novel information on the granulocyte proteome and offer insights into early, differentiated granulocyte reaction to stimuli, which contribute to a better understanding of molecular mechanisms involved in activation and recruitment of neutrophils, through inflammatory stimuli.

Anti-inflammatory effect of lanoconazole on 12-O-tetradecanoylphorbol-13-acetate- and 2,4,6-trinitrophenyl chloride-induced skin inflammation in mice.

SummaryBackgroundLanoconazole (LCZ) is a topical antifungal agent clinically used to treat fungal infections such as tinea pedis. LCZ has not only antifungal effects but also anti‐inflammatory effects, which have the potential to provide additional clinical benefits. However, the characteristic features of the inhibitory effects of LCZ on skin inflammation remain unclear.ObjectiveWe evaluated the inhibitory effects of topical application of LCZ, and compared the effects of LCZ with those of other antifungal agents including liranaftate, terbinafine and amorolfine.MethodsEach antifungal agent was topically applied on 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced irritant dermatitis and 2,4,6‐trinitrophenyl chloride‐induced contact dermatitis in mice (BALB/c). The ear thickness, myeloperoxidase activity and inflammatory mediator contents were evaluated.ResultsLCZ dose‐dependently suppressed 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced irritant dermatitis, suppressed the production of neutrophil chemotactic factors such as keratinocyte‐derived chemokine and macrophage inflammatory protein‐2, and inhibited neutrophil infiltration to the inflammation site. Moreover, 1% LCZ reduced the ear swelling in mice with 2,4,6‐trinitrophenyl chloride‐induced contact dermatitis in accordance with the inhibition of interferon‐γ production. The inhibitory potency of LCZ on these types of dermatitis in mice was stronger than that of other types of antifungal agents.ConclusionThe anti‐inflammatory effects of LCZ were exerted through the inhibition of inflammatory mediator production. These effects may contribute to the relief of dermatitis symptoms in patients with tinea pedis.

P13.09 Genomic analysis of paired IDHwt glioblastoma (GB) reveals recurrent alterations of MPDZ at relapse after radiotherapy and temozolomide (RTCT)

Abstract BACKGROUND GB are highly aggressive tumors which systematically relapse. Our objective was to identify disease progression mechanisms and genomic drivers of GB treatment resistance. MATERIAL AND METHODS Ten paired frozen tumors from initial and recurrent surgery after RTCT were screened by CGH Array. Next, NGS of the selected genes was performed on 19 paired tumors (38 samples). Molecular alterations were correlated with patient data. TCGA was used to characterize the molecular profile of MPDZ. RESULTS Nineteen IDHwt GB patients with a median age of 54.5 years (37.2–72.8) were included. Using CGH array, unsupervised analysis clustered the whole samples by paired of initial and recurrent tumors. However only 44% of CGH Array alterations were shared between initial and recurrent tumors (amplifications: 55%; deletions: 30%). The new alterations detected at relapse were amplifications in 25% and deletions in 23% of tumors. Two regions corresponding to 171 genes were lost at relapse (p=0.03): 19q13.33 and 19q13.41. Using DAVID genome, 3/171 genes (related to neutrophil chemotactic factors) were identified: FPR1, FPR2, FPR3. Moreover, 24 genes were lost (including MPDZ) and 2 genes were gained in 20% of recurrent tumors. Totally, 29 genes were analyzed by NGS and 4 genes showed pathogenic mutations shared by initial and recurrent tumors: FPR2, REL, TYRP1 and MPDZ. Only MPDZ showed, at relapse, an increasing rate of mutated variants and a new mutation affecting the splicing site. These alterations were independent from classical prognostic factors (age, sexe, karnofsky performans status, MMS and MGMT status) and from patient survivals. To explore MPDZ expression, we used TCGA initial dataset and observed that a lower RNA expression of MPDZ was associated with IDHwt (p&lt;0.001) and grade IV (p&lt;0.001) gliomas, reinforcing the potential pejorative impact of MPDZ loss. CONCLUSION Our results suggest that MPDZ is frequently altered at initial diagnosis with increased alterations in recurrent IDHwt GB after RTCT, suggesting that MPDZ impairment could contribute to the resistance/relapse mechanisms. Further investigations are needed to validate these results. Our results suggest that MPDZ is frequently altered at initial diagnosis with increased alterations in recurrent IDHwt GB after RTCT, suggesting that MPDZ impairment could contribute to the resistance/relapse mechanisms. Further investigations are needed to validate these results.