Biology subjects

Hajiaghayi, M.

Publications and source records attributed to Hajiaghayi, M..

3 recordsLinked to original sources

bioRxiv2024

Modulatory Effects of M3 Muscarinic Acetylcholine Receptor on Inflammatory Profiles of Human Memory T Helper Cells

Memory T helper (Th) cells, generated after immunogenic challenge, are crucial in directing the adaptive immune response. Muscarinic ACh receptor (mAChR) subtypes expressed by immune cells can be stimulated with acetylcholine or muscarinic-selective drug oxotremorine-M. Cholinergic signaling can influence immune cells, but it is not known how cholinergic stimuli regulate memory Th cells. This study focused on the role of mAChRs, specifically the M3 muscarinic ACh receptor (M3R), in the cytokine profile and NF-{kappa}B p65 activity of primary human memory Th cells. Memory Th cells (CD3+CD4+CD45RA-CD45RO+) were isolated from healthy participants peripheral blood. Cell culture was performed with anti-CD3/anti-CD28/anti-CD2 reagent, oxotremorine-M (M1R-M5R agonist), atropine (M1R-M5R antagonist), and J104129 (M3R-selective antagonist). MR1-MR5 genes CHRM1-CHRM5 were measured with RT-qPCR. Protein expression of M3R and phosphorylated NF-{kappa}B p65 were quantified by Western blot. The secretion of IFN-{gamma}, IL-17A, and IL-4 was assessed by ELISA and intracellular cytokine staining flow cytometry. CHRM3, encoding M3R, was knocked out using CRISPR-Cas9 gene targeting. Memory Th cells expressed all five mAChR subtypes. Oxotremorine-M increased IFN-{gamma} and IL-17A while reducing IL-4 in an atropine-sensitive manner. Stimulation of mAChRs in cells with CHRM3-knockout or M3R blockade prevented increases in IFN-{gamma} and IL-17A but continued to inhibit IL-4. mAChR stimulation enhanced NF-{kappa}B p65 activity without affecting cell proliferation, viability, or M3R expression. This investigation demonstrates that muscarinic signaling increases the pro-inflammatory profile of memory Th cells, including NF-{kappa}B p65, IFN-{gamma}, and IL-17A, with a reduction in IL-4. Focusing on M3R blockers could modulate adaptive immune responses and alleviate immune-related conditions.

immunology↗

bioRxiv2024

β2-Adrenergic Biased Agonist Inhibits the Development of Th17 and the Response of Memory Th17 Cells in an NF-κB-Dependent Manner.

IntroductionAdrenergic receptors regulate metabolic, cardiovascular and immunological functions in response to the sympathetic nervous system. The effect of {beta}2-adrenergic receptor (AR) as a high-expression receptor on different subpopulations of T cells is complex and varies depending on the type of ligand and context. While traditional {beta}2-AR agonists generally suppress T cells, they potentially enhance IL-17A production by Th17 cells. The effects of pharmacological drugs that count as biased agonists of AR like nebivolol are not completely understood. We investigated the impact of nebivolol on human memory CD4+ T (Th1, Th2, Th17) cells and polarized naive Th17 cells highlighting its potential for IL-17A suppression via a non-canonical {beta}2AR cell-signaling pathway. MethodsThe effects of nebivolol were tested on healthy human peripheral blood mononuclear cells, purified memory Th cells, and polarized naive Th17 cells activated with antiCD3/antiCD28/antiCD2 ImmunoCult reagent. IFN-{gamma}, IL-4, and IL-17A which are primarily derived from Th1, Th2, and Th17 cells respectively, were quantified by ELISA and flow cytometry. IL-10 was measured by ELISA. Gene expression of RORC, ADRB1, ADRB2, and ADRB3 was evaluated by qPCR. The ADRB2 gene was knocked out in memory Th cells using CRISPR/Cas9. Protein expression of phosphorylated-serine133-CREB and phosphorylated-NF-{kappa}B p65 was assessed by Western blot. Proliferation was assessed by fluorescent dye loading and flow cytometry. ResultsNebivolol treatment decreased IL-17A and IFN-{gamma} secretion by activated-memory Th cells and elevated IL-4 levels. Nebivolol reduced the proportion of IL-17A+ Th cells and downregulated RORC expression. Unlike the {beta}2-AR agonist terbutaline, nebivolol inhibited the shift of naive CD4+ T cells towards the Th17 phenotype. IL-10 and proliferation index remained unchanged. Nebivolol-treated {beta}2-knockout memory Th cells showed significant inhibition of {beta}2AR-mediated signaling, evidenced by the absence of IL-17A suppression compared to controls. Phosphorylation of the NF-{kappa}B p65 subunit was inhibited by nebivolol, but CREB phosphorylation was not changed, suggesting a selective transcriptional control. ConclusionsThe findings demonstrate that nebivolol acts through a {beta}2-AR-mediated signaling pathway, as a distinctive anti-inflammatory agent capable of selectively shifting Th17 cells and suppressing phosphorylation of NF-{kappa}B. This highlights nebivolols potential for therapeutic interventions in chronic autoimmune conditions with elevated IL-17A levels.

immunology↗

bioRxiv2024

A Digital Microfluidic Platform for the Microscale Production of Functional Immune Cell Therapies

Genetically engineering human immune cells has been shown to be an effective approach for developing novel cellular therapies to treat a wide range of diseases. To expand the scope of these cellular therapies while solving persistent challenges, extensive research and development is still required. Electroporation has recently emerged as one of the most popular techniques for inserting biological payloads into human immune cells to perform genetic engineering. However, several recent studies have reported that electroporation can negatively impact cell functionality. Additionally, the requirement to use large amounts of cells and expensive payloads to achieve efficient delivery can drive up the costs of development efforts. Here we use a digital microfluidic enabled electroporation system (referred to as triDrop) and compare them against two state-of-the-art commercially available systems for the engineering of human T cells. We describe the ability to use triDrop for highly viable, highly efficient transfection while using substantially fewer cells and payload. Subsequently, we perform transcriptomic analysis on cells engineered with each of the three systems and show that electroporation with triDrop lead to less dysregulation of several functionally relevant pathways. Finally, in a direct comparison of immunotherapeutic functionality, we show that T cells engineered with triDrop have an improved ability to mount an immune response when presented with tumor cells. These results show that the triDrop platform is uniquely suited to produce functionally engineered immune cells while also reducing the costs of cell engineering compared to other commercially available systems.

bioengineering↗