Several studies have reported that the host-microbe interactions in the gut modulate the host serotonin or 5-hydroxytryptamine (5-HT) system. Here, we evaluated the effects ofAkkermansia muciniphilaand its extracellular vesicles (EVs) on genes pertaining to the serotonergic system in the colon and hippocampus of mice. Male C57BL/6J mice were administered viableA. muciniphilaand its EVs for 4 weeks. The serotonin levels in the colon, hippocampus, and serum of mice, as well as the human colon carcinoma cells (Caco-2), were measured by ELISA assays. Also, the effects ofA. muciniphilaand its EVs on the expression of serotonin system genes in the colon and hippocampus were examined.A. muciniphilaand its EVs may have a biological effect on the induction of serotonin levels in the colon and hippocampus of mice. Also, EVs increased the serotonin level in the Caco-2 cell line. In contrast, both treatments decreased the serotonin level in the serum. Both the bacterium and its EVs had significant effects on the mRNA expression of genes, involved in serotonin signaling/metabolism in the colon and hippocampus of mice. Moreover,A. muciniphilaand its EVs affected the mRNA expression of inflammatory cytokines (Il-10andTnf-α) in the colon, however, there is no significant difference in inflammatory cell infiltrate in the histopathology of the colon. The presence ofA. muciniphilaand its EVs in the gut promotes serotonin concentration, they also affect serotonin signaling/metabolism through the gut-brain axis and may be considered in new therapeutic strategies to ameliorate serotonin-related disorders.

We assessed effect of Akkermansia muciniphila and its extracellular vesicles on toll-like receptors and tight junction expression in human epithelial colorectal adenocarcinoma cells (Caco-2). The intestinal microbiota plays an important role in the intestinal homeostasis through its metabolites and derivatives. Interacting with immune cells and intestinal epithelial pattern recognition receptors (PRRs), intestinal microbiota regulates the function of the digestive barrier and inflammation caused by the metabolic diseases. A. muciniphila was cultured on a mucin-containing medium and its EVs was extracted by ultracentrifugation. This bacterium was treated in the MOI=10 and its EVs at the concentrations of 0.1, 0.5 and 5 µg on Caco-2 cells. After 24 hours, the expression of tight junction and toll-like receptor genes were investigated by quantitative real time PCR method. A. muciniphila increased the expression of tlr2 and tlr4. However, EVs at all of the concentrations showed a decrease in tlr4 expression. EVs at the concentrations of 0.1 and 0.5 µg/ml decreased the expression of tlr2. A. muciniphila significantly increased the expression of ocldn and cldn4. Both this bacterium and EVs increased the expression of zo2 in the cell line. Furthermore, this data show that A. muciniphila derived EVs have a dose-independent effect on Caco-2 cells. This preliminary research shows A. muciniphila and its EVs both may increase the integrity of the intestinal barrier. A. muciniphila derived EVs also reduces the inflammation so that EVs of this bacterium can be used as an appropriate target for the treatment of metabolic syndrome and inflammatory bowel diseases.

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG–treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-β concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells. In Iran, cutaneous leishmaniasis (CL) is a widespread and highly endemic disease in young individuals. To date, treatment strategy is based on chemotherapy accompanied with high incidence of toxicity and drug resistance. Distinctive mode of action of defensins (members of antimicrobial peptides) with low susceptibility to resistance and low toxicity to mammalian cells makes them suitable candidates for anti-leishmanial agents. The most active human defensin is human neutrophil peptide-1 (HNP-1) produced by neutrophils; the first effector cells during Leishmania infection. In this work, we used recombinant HNP-1 (rHNP-1) against both the promastigote and amastigote forms of Leishmania (L.) major. Furthermore, immunomodulatory effect of rHNP-1 on Leishmania-infected neutrophils was investigated. Our result showed that rHNP-1 has anti-parasitic effect against L. major promastigotes and amastigotes and also reduces infectivity rate of Leishmania-infected neutrophils. Moreover, assessment of cytokine production from Leishmania-infected neutrophils reveals an increase in TNF-α and a decrease in TGF-β production after rHNP-1 treatment; a cytokine pattern anticipated to facilitate control of parasites. The immunomodulatory effect of rHNP-1 on cytokine production from parasite-infected neutrophils besides its direct effect on free parasites is considered as promising step towards developing new anti-leishmanial agents.

Objective: Gastrointestinal (GI) tract, like other mucosal surface, is colonized with a microbial population known as gut microbiota. Outer membrane vesicles (OMVs) which are produced by gram negative bacteria could be sensed by Toll like receptors (TLRs). The interaction between gut microbiota and TLRs affects homeostasis and immune responses. In this study, we evaluated TLR2, TLR4 genes expression and cytokines concentration in Caco-2 cell line treated with Bacteroides fragilis (B. fragilis) and its OMVs. Materials and Methods: In this experimental study, OMVs were extracted using sequential centrifugation and their physicochemical properties were evaluated as part of quality control assessment. Caco-2 cells were treated with B. fragilis and its OMVs (180 and 350 mu g/ml). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to assess TLR2 and TLR4 mRNA expression levels. Pro-inflammatory (IFN gamma) and anti-inflammatory (IL-4 and IL-10) cytokines were evaluated by ELISA. Results: B. fragilis significantly decreased TLR2 and slightly increased TLR4 mRNA levels in Caco-2 cell line. The TLR2 mRNA level was slightly increased at 180 and 350 mu g/ml of OMVs. Conversely, the TLR4 mRNA level was decreased at 180 mu g/ml of OMVs, while it was significantly increased at 350 mu g/ml of OMVs. Furthermore, B. fragilis and its OMVs significantly increased and decreased IFN gamma concentration, respectively. Anti-inflammatory cytokines were increased by B. fragilis and its OMVs. Conclusion: B. fragilis and its OMVs have pivotal role in the cross talk between gut microbiota and the host especially in the modulation of the immune system. Based on the last studies on immunomodulatory effect of B. fragilis derived OMVs on immune cells and our results, we postulate that B. fragilis derived OMVs could be possible candidates for the reduction of immune responses.