Oropharyngeal Carriage of hpl-Containing Haemophilus haemolyticus Predicts Lower Prevalence and Density of NTHi Colonisation in Healthy Adults.

Nontypeable Haemophilus influenzae (NTHi) is a major respiratory pathogen that initiates infection by colonising the upper airways. Strategies that interfere with this interaction may therefore have a clinically significant impact on the ability of NTHi to cause disease. We have previously shown that strains of the commensal bacterium Haemophilus haemolyticus (Hh) that produce a novel haem-binding protein, haemophilin, can prevent NTHi growth and interactions with host cells in vitro. We hypothesized that natural pharyngeal carriage of Hh strains with the hpl open reading frame (Hh-hpl+) would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Oropharyngeal swabs were collected from 257 healthy adults in Australia between 2018 and 2019. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl- genotype. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was assessed in individuals that carried either Hh-hpl- (n = 25) or Hh-hpl+ (n = 25). Compared to carriage of Hh-hpl- strains, adult (18-65 years) and elderly (>65 years) participants that were colonised with Hh-hpl+ were 2.43 or 2.67 times less likely to carry NTHi in their oropharynx, respectively. Colonisation with high densities of Hh-hpl+ correlated with a low NTHi carriage load and a 2.63 times lower likelihood of acquiring/maintaining NTHi colonisation status between visits. Together with supporting in vitro studies, these results encourage further investigation into the potential use of Hh-hpl+ as a respiratory probiotic candidate for the prevention of NTHi infection.

Haemophilin-Producing Strains of Haemophilus haemolyticus Protect Respiratory Epithelia from NTHi Colonisation and Internalisation.

Nontypeable Haemophilus influenzae (NTHi) is a significant respiratory tract pathogen responsible for infections that collectively pose a substantial health and socioeconomic burden. The clinical course of these infections is largely dictated by NTHi interactions with host respiratory epithelia, and thus, approaches that disrupt colonisation and invasion may have significant therapeutic potential. Survival, successful host-cell interactions, and pathogenesis are reliant on NTHi's ability to sequester host-derived haem. Previously, we demonstrated the therapeutic potential of exploiting this haem-dependence using a closely related competitor bacterium, Haemophilus haemolyticus (Hh). Hh strains capable of producing the novel haem-binding protein haemophilin (Hpl) possessed potent inhibitory activity by restricting NTHi access to haem in a broth co-culture environment. Here, we extend this work to cell culture models that more closely represent the human respiratory epithelium and show that Hh strains with high levels of hpl expression protect epithelial cell line monolayers against adhesion and invasion by NTHi. Inhibitory activity was dependent on the level of Hpl production, which was stimulated by NTHi challenge and nasopharyngeal cell exposure. Provided these protective benefits translate to in vivo applications, Hpl-producing Hh may have probiotic utility against NTHi infections by inhibiting requisite nasopharyngeal colonisation.

Streptococcus Thermophilus UASt-09 Upregulates Goblet Cell Activity in Colonic Epithelial Cells to a Greater Degree than other Probiotic Strains.

Probiotics have been widely used in maintaining gastrointestinal health, despite their actual mechanism remaining obscure. There are several hypotheses behind the beneficial effects of probiotics including the regulation of intestinal barrier function and improvement in immune responses in the gastrointestinal system. Multiple probiotics have been introduced in the market as effective dietary supplements in improving gastrointestinal integrity, but there are no or few studies that demonstrate their underlying mechanism. In the current study, we investigated and compared the efficacy of four probiotics (based on different bacterial species) in refining gastrointestinal health by improving mucus biosynthesis and intestinal immune response under in-vitro conditions. By analyzing the gene expression of mucus biosynthesis and intestinal immune response markers, we found that probiotic Streptococcus thermophilus UASt-09 showed promising potential in refining mucosal barrier and gastrointestinal health in human colonic epithelial cells, as compared to other commercial probiotics.

In Vitro Anti-NTHi Activity of Haemophilin-Producing Strains of Haemophilus haemolyticus.

Nontypeable Haemophilus influenzae (NTHi) is a leading causative organism of opportunistic respiratory tract infections. However, there are currently no effective vaccination strategies, and existing treatments are compromised by antibiotic resistance. We previously characterized Haemophilus haemolyticus (Hh) strains capable of producing haemophilin (HPL), a heme-binding protein that restricts NTHi growth by limiting its access to an essential growth factor, heme. Thus, these strains may have utility as a probiotic therapy against NTHi infection by limiting colonization, migration and subsequent infection in susceptible individuals. Here, we assess the preliminary feasibility of this approach by direct in vitro competition assays between NTHi and Hh strains with varying capacity to produce HPL. Subsequent changes in NTHi growth rate and fitness, in conjunction with HPL expression analysis, were employed to assess the NTHi-inhibitory capacity of Hh strains. HPL-producing strains of Hh not only outcompeted NTHi during short-term and extended co-culture, but also demonstrated a growth advantage compared with Hh strains unable to produce the protein. Additionally, HPL expression levels during competition correlated with the NTHi-inhibitory phenotype. HPL-producing strains of Hh demonstrate significant probiotic potential against NTHi colonization in the upper respiratory tract, however, further investigations are warranted to demonstrate a range of other characteristics that would support the eventual development of a probiotic.

NLRP3-Dependent and -Independent Processing of Interleukin (IL)-1ß in Active Ulcerative Colitis.

A contributing factor in the development of ulcerative colitis (UC) and Crohn's disease (CD) is the disruption of innate and adaptive signaling pathways due to aberrant cytokine production. The cytokine, interleukin (IL)-1ß, is highly inflammatory and its production is tightly regulated through transcriptional control and both inflammasome-dependent and inflammasome- independent proteolytic cleavage. In this study, qRT-PCR, immunohistochemistry, immunofluorescence confocal microscopy were used to (1) assess the mRNA expression of NLRP3, IL-1ß, CASP1 and ASC in paired biopsies from UC and CD patient, and (2) the colonic localization and spatial relationship of NLRP3 and IL-1ß in active and quiescent disease. NLRP3 and IL-1ß were found to be upregulated in active UC and CD. During active disease, IL-1ß was localized to the infiltrate of lamina propria immune cells, which contrasts with the near-exclusive epithelial cell layer expression during non-inflammatory conditions. In active disease, NLRP3 was consistently expressed within the neutrophils and other immune cells of the lamina propria and absent from the epithelial cell layer. The disparity in spatial localization of IL-1ß and NLRP3, observed only in active UC, which is characterized by a neutrophil-dominated lamina propria cell population, implies inflammasome-independent processing of IL-1ß. Consistent with other acute inflammatory conditions, these results suggest that blocking both caspase-1 and neutrophil-derived serine proteases may provide an additional therapeutic option for treating active UC.

TRPV1 mediates capsaicin-stimulated metabolic activity but not cell death or inhibition of interleukin-1ß release in human THP-1 monocytes.

Human monocytes and dendritic cells express transient receptor potential vanilloid 1 (TRPV1) which may play a role in mediating the inflammatory, immune and cancer surveillance responses of these cells. The aim of the present study was to investigate TRPV1 expression and function in THP-1 monocytic cells. RT-PCR and Western blot were used to detect TRPV1. The metabolic activity and viability of THP-1 cells following exposure to vanilloids was assessed using resorufin production from rezazurin. Cytokine release was measured using ELISA. TRPV1 was expressed in cultured THP-1 monocytic cells and naïve monocytes. Lower concentrations (<250 µM) of capsaicin, but not other putative TRPV1 agonists, were shown to stimulate cell metabolic activity, whereas at concentrations >250 µM, all agonists decreased metabolic activity. Capsaicin-stimulated THP-1 metabolic activity was blocked by the TRPV1 antagonist, 5-iodo-resiniferatoxin (5'-IRTX), whereas the decline in resorufin production by THP-1 cells at higher capsaicin concentrations (due to cell death), was not affected by 5'-IRTX. Finally, capsaicin (≤125 µM) significantly increased lipopolysaccharide-stimulated IL-6 and TNF-α release from THP-1 cells, whereas phytohaemagglutinin-stimulated IL-1ß, TNF-α, MCP-1 and IL-6 release were concentration-dependently inhibited by capsaicin. Modulation of IL-1ß release was not TRPV1 mediated. Overall, these results show that functional TRPV1 channels are present in naïve monocytes and THP-1 cells, and when activated, increase cell metabolic activity. In addition, capsaicin modifies cytokine release from THP-1 cells and induces cell death, most likely by a mechanism that is independent of TRPV1 activation.

MCC950, a specific small molecule inhibitor of NLRP3 inflammasome attenuates colonic inflammation in spontaneous colitis mice.

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1ß, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1ß release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

Regulation and Sensing of Inflammasomes and Their Impact on Intestinal Health.

Pattern recognition receptors such as nucleotide-binding oligomerization domain (NOD)-containing protein receptors (NLRs) and the pyrin and hematopoitic interferon-inducible nuclear protein (HIN) domain (PYHIN) receptors initiate the inflammatory response following cell stress or pathogenic challenge. When activated, some of these receptors oligomerize to form the structural backbone of a signalling platform known as an inflammasome. Inflammasomes promote the activation of caspase-1 and the maturation of the proinflammatory cytokines, interleukin (IL)-1ß and IL-18. The gut dysregulation of the inflammasome complex is thought to be a contributing factor in the development of inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn's disease (CD). The importance of inflammasomes to intestinal health has been emphasized by various inflammasome-deficient mice in dextran sulphate sodium (DSS) models of intestinal inflammation and by the identification of novel potential candidate genes in population-based human studies. In this review, we summarise the most recent findings with regard to the formation, sensing, and regulation of the inflammasome complex and highlight their importance in maintaining intestinal health.

Expression of acquired macrolide resistance genes in Haemophilus influenzae.

OBJECTIVES: To investigate the phenotypic effect of expression of selected acquired macrolide resistance genes (AMRGs) in non-typeable Haemophilus influenzae (NTHi). METHODS: The AMRGs erm(A), erm(B) and erm(C) were cloned into Escherichia coli JM109 using the shuttle vector pLS88; constructed plasmids extracted from suitable clones were used to transform H. influenzae Rd by electroporation. Erythromycin and azithromycin MICs for suitable transformants were determined by broth microdilution. AMRG expression was determined using quantitative PCR on transformant cDNA with locked nucleic acid dual-labelled hydrolysis probes. RESULTS: Expression of all AMRGs was observed in the transformants. Some variation in expression between the AMRGs was apparent, but expression of all genes was associated with a notable increase in erythromycin and azithromycin MICs compared with untransformed H. influenzae Rd. CONCLUSIONS: While the establishment of erm genes within WT populations of NTHi remains contentious, H. influenzae is capable of expression of erm. Expression may be associated with a subsequent decreased susceptibility to macrolides in isolates and future monitoring of these genes in NTHi isolates is warranted.

ERBB3: A potential serum biomarker for early detection and therapeutic target for devil facial tumour 1 (DFT1).

Devil Facial Tumour 1 (DFT1) is one of two transmissible neoplasms of Tasmanian devils (Sarcophilus harrisii) predominantly affecting their facial regions. DFT1's cellular origin is that of Schwann cell lineage where lesions are evident macroscopically late in the disease. Conversely, the pre-clinical timeframe from cellular transmission to appearance of DFT1 remains uncertain demonstrating the importance of an effective pre-clinical biomarker. We show that ERBB3, a marker expressed normally by the developing neural crest and Schwann cells, is immunohistohemically expressed by DFT1, therefore the potential of ERBB3 as a biomarker was explored. Under the hypothesis that serum ERBB3 levels may increase as DFT1 invades local and distant tissues our pilot study determined serum ERBB3 levels in normal Tasmanian devils and Tasmanian devils with DFT1. Compared to the baseline serum ERBB3 levels in unaffected Tasmanian devils, Tasmanian devils with DFT1 showed significant elevation of serum ERBB3 levels. Interestingly Tasmanian devils with cutaneous lymphoma (CL) also showed elevation of serum ERBB3 levels when compared to the baseline serum levels of Tasmanian devils without DFT1. Thus, elevated serum ERBB3 levels in otherwise healthy looking devils could predict possible DFT1 or CL in captive or wild devil populations and would have implications on the management, welfare and survival of Tasmanian devils. ERBB3 is also a therapeutic target and therefore the potential exists to consider modes of administration that may eradicate DFT1 from the wild.

NLRP3 Inhibitors as Potential Therapeutic Agents for Treatment of Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a group of intestinal disorders characterised by chronic relapsing inflammation of the small intestine and colon. IBD manifests as either ulcerative colitis or Crohn's disease and increases the risk of developing colorectal cancer. METHODS: Here, we have reviewed current treatment regimen for IBD utilising anti-inflammatory drugs, immune system suppressors and antibiotics or a combination of these. However, these therapeutics lead to a number of adverse effects, remission or significant non-responsiveness creating an urgent need to develop potent drugs with novel mechanisms of action for IBD. RESULTS: The inflammasome is a multiprotein complex that assembles to a key innate immune system signalling platform and is involved in the pathogenesis of inflammatory and autoimmune diseases. A number of investigations show that the NLRP3 inflammasome recognizes microbial and cell stress components and serve as a platform for caspase-1 activation and pro-inflammatory cytokine IL-1ß, IL18 maturation. Although the exact aetiology of IBD is unknown, uncontrolled NLRP3 Inflammasome activation has shown to play a major role in the chronic intestinal inflammation and mature IL-1ß and IL18 are consistently associated with increased colitis and colitis associated colorectal cancer development. CONCLUSION: In this review, we discuss the experimental NLRP3 inhibitors that have been investigated in IBD experimental models. The potential mechanism of action of these inhibitors such as inhibiting NF-κB activation and decreasing mitochondrial reactive oxygen species are discussed in detail. We further expand the controversial role of NLRP3 in IBD and future issues that might arise from the long term use of NLRP3 inhibitors in IBD therapy.

Bitter melon protects against ER stress in LS174T colonic epithelial cells.

BACKGROUND: Bitter Melon (BM) has been used as a functional food in traditional Chinese and Indian medicine for many generations and has gained a great deal of attention due to its apparent benefits in moderating some of the pathogenic processes in a variety of inflammatory conditions. BM extract (BME) has been shown to possess strong anti-oxidant properties. In addition, it can ameliorate oxidative stress and potentially ER stress. There is increasing evidence that oxidative and ER stress are major contributors for intestinal secretory cell dysfunction which leads to local inflammation and disease pathogenesis that are hallmarks of inflammatory bowel diseases (IBD). Hence, the search for potential therapeutics against ER stress and oxidative stress in intestinal epithelial secretory cells may provide valuable resources for the management of IBD. The aim of the present study was to investigate the effects of BME in ameliorating ER stress in colonic epithelial cells. METHODS: Human colonic adenocarcinoma LS174T cells were used for the assessment of BME effects on colonic epithelial cells in vitro. Cell viability was assessed using trypan blue exclusion and the effect of BME in ameliorating tunicamycin (TM)-induced ER stress was determined by analysing the mRNA expression of the common ER stress markers; ATF6, XBP1, GRP78, CHOP and PERK by quantitative RT-PCR and GRP78 and CHOP by western blot. RESULTS: In the absence of ER stress, BME exhibited no cell toxicity up to 2.0% w/v and no significant effect on the basal mRNA expression of ER stress markers in LS174T cells. In contrast, pre-treatment of LS174T cells with BME followed by induction of ER stress resulted in a significant decrease in mRNA expression of ATF6, XBP1, GRP78, CHOP and PERK and protein expression of GRP78 and CHOP. Co-treatment during induction of ER stress and post- treatment following induction of ER Stress in LS174T cells resulted in a lower but still significant reduction in mRNA expression levels of most ER stress markers. CONCLUSIONS: This is one of the first studies demonstrating the efficacy of BME in reducing expression of ER stress markers in colonic epithelial cells suggesting the potential of BME as a dietary intervention in ameliorating ER stress and oxidation in IBD. Interestingly, while the most significant effect was seen with pre-treatment of cells with BME there was a reduced but still significant effect when co-treated or even post-treated. This suggests that BME may even be effective in modulating ER stress in the face of an existing cell stress environment.

Dendritic Cells Modulate Iron Homeostasis and Inflammatory Abilities Following Quercetin Exposure.

BACKGROUND: Fruits and vegetables are rich in plant polyphenols, whose consumption is encouraged in healthy dietary regimes due to their antioxidants and anti-inflammatory effects. These organic molecules exhibit numerous properties including phylochelation; the ability to complex metal ions, including highly reactive iron. Among polyphenols, we focused our attention on quercetin that previously demonstrated its ability to reduce dendritic cells (DCs) inflammatory cytokine secretion and antigen presentation following LPS exposure. Dendritic cell inflammatory response is also associated with modulation of several iron metabolism related genes. OBJECTIVE: To characterize the axis between quercetin exposure and iron extracellular transport that may explain polyphenol anti-inflammatory abilities. METHOD: Bone marrow derived DCs were exposed to 25µM of quercetin on day 7 and treated with 1 µg/mL of LPS on day 8. The relation between quercetin exposure and the expression level of genes involved in iron homeostasis was addressed by qPCR. The axis between iron export and quercetin exposure was confirmed in vitro and in vivo using quercetin gavage and quercetin-enriched diet. RESULTS: Here we demonstrate that DCs, exposed to quercetin, activate a pattern of genes that increase extracellular iron export, resulting in an overall decrease in the intracellular iron content and consequent diminished inflammatory abilities. This DCs phenotype is consistent with anti-inflammatory phenotype of the mucosal resident DCs, the ones most commonly exposed to polyphenols. CONCLUSIONS: Iron balance is a crucial checkpoint for DCs inflammatory abilities. Quercetin-enriched nutritional regimes that favor DCs extracellular iron transport could reduce the incidence of chronic inflammatory syndromes.

Characterisation of colonic dysplasia-like epithelial atypia in murine colitis.

AIM: To determine if exacerbation of pre-existing chronic colitis in Winnie (Muc2 mutant) mice induces colonic dysplasia. METHODS: Winnie mice and C57BL6 as a genotype control, were administered 1% w/v dextran sulphate sodium (DSS) orally, followed by drinking water alone in week-long cycles for a total of three cycles. After the third cycle, mice were killed and colonic tissue collected for histological and immunohistochemical evaluation. Inflammation and severity of dysplasia in the colonic mucosa were assessed in H&E sections of the colon. Epithelial cell proliferation was assessed using Ki67 and aberrant ß-catenin signalling assessed with enzyme-based immunohistochemistry. Extracted RNA from colonic segments was used for the analysis of gene expression using real-time quantitative PCR. Finally, the distribution of Cxcl5 was visualised using immunohistochemistry. RESULTS: Compared to controls, Winnie mice exposed to three cycles of DSS displayed inflammation mostly confined to the distal-mid colon with extensive mucosal hyperplasia and regenerative atypia resembling epithelial dysplasia. Dysplasia-like changes were observed in 100% of Winnie mice exposed to DSS, with 55% of these animals displaying changes similar to high-grade dysplasia, whereas high-grade changes were absent in wild-type mice. Occasional penetration of the muscularis mucosae by atypical crypts was observed in 27% of Winnie mice after DSS. Atypical crypts however displayed no evidence of oncogenic nuclear ß-catenin accumulation, regardless of histological severity. Expression of Cav1, Trp53 was differentially regulated in the distal colon of Winnie relative to wild-type mice. Expression of Myc and Ccl5 was increased by DSS treatment in Winnie only. Furthermore, increased Ccl5 expression correlated with increased complexity in abnormal crypts. While no overall difference in Cxcl5 mucosal expression was observed between treatment groups, epithelial Cxcl5 protein appeared to be diminished in the atypical epithelium. CONCLUSION: Alterations to the expression of Cav1, Ccl5, Myc and Trp53 in the chronically inflamed Winnie colon may influence the transition to dysplasia.

Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFα secretion.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that in response to microbial infections generate long-lasting adaptive immune response. Following microbial uptake, DCs undergo a cascade of cellular differentiation that ultimately leads to "mature" DCs. Mature DCs produce a variety of inflammatory cytokines, including tumor necrosis factor-α (TNFß) a key cytokine for the inflammatory cascade. In numerous studies, polyphenols, including quercetin, demonstrated their ability to suppress TNFα secretion and protect from the onset of chronic inflammatory disorders. We show that murine bone marrow derived DCs express Slpi following quercetin exposure. Slpi is known to suppress LPS mediated NFκB activation, thus, it was hypothesized that its expression could be the key step for polyphenol induced inflammatory suppression. Slpi-KO DCs poorly respond to quercetin administration failing to reduce TNFα secretion in response to quercetin exposure. Supernatant from quercetin exposed DCs could also reduce LPS-mediated TNFα secretion by unrelated DCs, but this property is lost using an anti-Slpi antibody. In vivo, oral administration of quercetin is able to induce Slpi expression. Human biopsies from inflamed tract of the intestine reveal the presence of numerous SLPI+ cells and the expression level could be further increased by quercetin administration. We propose that quercetin induces Slpi expression that in turn reduces the inflammatory response. Our data encourages the development of nutritional strategies to improve the efficiency of current therapies for intestinal chronic inflammatory syndrome and reduce the risks of colorectal cancer development.

Effect of epithelial cell type on in vitro invasion of non-typeable Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) have been shown to have variable ability for in vitro invasion with a range of epithelial cells, and increased invasion of BEAS-2B cells has been associated with altered penicillin binding protein3 (PBP3), which is concerning as these strains are increasing worldwide. The aim of the study was to investigate the effect of respiratory cell type and the presence of altered PBP3 on the in vitro invasion of NTHi. A collection of 16 clinical NTHi isolates was established, 7 had normal PBP3, and 9 had altered PBP3 as defined by an N526K substitution. The isolates were tested for invasion of BEAS-2B, NHBE, A549 and NCI-H292 respiratory epithelial cells in vitro using a gentamicin survival assay, with invasion measured as the percentage of intracellular organisms relative to the initial inoculum. The overall median invasion for the 16 NTHi isolates for cell types BEAS-2B, NHBE, A549 and NCI-H292 cells were 3.17, 2.31, 0.11 and 1.52 respectively. The differences were statistically significant for BEAS-2B compared to A549 (P=0.015) and A549 compared to NCI-H292 (P=0.015), and there were also very marked differences in invasion for some individual isolates depending on the cell type used. There was a consistent bias for invasion of isolates with normal versus abnormal PBP3: and this was statistically significant for BEAS-2B (0.07 to 9.90, P=0.031) and A549 cells (0.02 to 1.68, P=0.037). These results show that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the cell line used, and that the association between altered PBP3 and increased invasion is conserved across multiple cell lines.

Capsaicin-Induced Death of Human Haematological Malignant Cell Lines Is Independent of TRPV1 Activation.

The effect of the plant-derived vanilloid, capsaicin (CAP), on the metabolic activity of THP-1, U266B1 and U937 hematological malignancy cells was determined. CAP reduced metabolic activity in a concentration-dependent manner in the three cell lines. A biphasic effect was observed on THP-1 cells (EC50: IC50 (95% CI) 32.9 (19.9-54.3)/219 (144-246) µmol/l). U266B1 cells were more resistant to CAP than THP-1 and U937. Metabolic activity was significantly inhibited by CAP in U937 compared to U266B1 cells (IC50: 197 versus 431 µmol/l, respectively, p < 0.008). Transient receptor potential vanilloid-1 (TRPV1) and CB1 antagonists (SB452533 and AM251, respectively) suppressed the CAP-induced increase in THP-1 cell metabolic activity (p < 0.001). AM251 and SB452533 appeared to act as partial agonists and displayed a synergistic effect with CAP in U937 cells. CAP inhibits the metabolic activity of malignant hematological cells through non-TRPV1-dependent mechanisms.