Characterization of prostanoids response to Bordetella pertussis antigen BscF and Tdap in LPS-challenged monocytes.

Prostanoids are potent inflammatory mediators that play a regulatory role in the innate immune activation of the adaptive immune response to determine the duration of protection against infection. We aim to quantify the modulation of prostanoids profiles in lipopolysaccharide (LPS)-stimulated THP-1 cells treated with the novel pertussis antigen BscF. We compared the effect with pertussis antigens present in the current Tdap vaccine to understand the immunomodulatory effect that might contribute to the diminished Tdap vaccine effectiveness. The inflammatory challenge with LPS induced a robust elevation of most prostanoid family members compared to the control treatment. Treatment with BscF and Tdap significantly reduced the LPS-stimulated elevation of prostaglandins (PGs) D2, E2, and F2α, as well as thromboxane (TX) A2 levels. An opposite trend was observed for PGI2, as both antigens accelerated the LPS-stimulated upregulation. Further, we quantified cyclooxygenases (COXs) that catalyze the biosynthesis of prostanoids and found that both antigens significantly reduced LPS-stimulated COX-1 and COX-2, demonstrating that the waning of acellular pertussis vaccines' protective immunity may be due to other downstream enzymes not related to COXs. Our present study validates the potential role of BscF as an adjuvant, resulting in the next-generation pertussis vaccine discovery.

Modulation of Inflammatory Signaling Molecules in Bordetella pertussis Antigen-Challenged Human Monocytes in Presence of Adrenergic Agonists.

BscF is a type III secretion system (T3SS) needle protein from Bordetella pertussis and has previously been shown to induce a sufficient Th1 and Th17 response in human monocytes and mice as a prerequisite for long-lasting protective immunity against pertussis infection. In our current study, we aim to compare the modulation of inflammatory signaling molecules as a direct measure of the immune response to the B. pertussis antigens BscF and Tdap in the presence or absence of the adrenergic receptor agonists phenylephrine (PE) or isoproterenol (ISO) to observe differences that may contribute to the diminished protective immunity of the current acellular pertussis (aP) vaccine, Tdap. Stimulation of human monocyte THP-1 cells with LPS, BscF, and Tdap induced a robust elevation of CCL20, CXCL10, PGE2, and PGF2α among most chemokine and prostanoid members when compared with the control treatment. Treatment with the adrenergic agonist PE or ISO significantly enhanced the BscF- and Tdap-stimulated modulation of CCL20 and CXCL10 but not PGE2 and PGF2α, suggesting that adrenergic modulation of pertussis antigen responses might be a new therapeutic strategy to improve the longevity of pertussis immunity. Stimulation of THP-1 cells with BscF alone initiated significant expression of CXCL10 and PGF2α but not when Tdap was used, suggesting that BscF might be an important pertussis antigen for next-generation pertussis vaccines or when combined with the current aP vaccine. Our data offer opportunities for designing new therapeutic approaches against pertussis infection.

Method of Microglial DNA-RNA Purification from a Single Brain of an Adult Mouse.

Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population.