Human Lung Mast Cells as a Possible Reservoir for Coronavirus: A Novel Unrecognized Mechanism for SARS-CoV-2 Immune-Mediated Pathology.

The pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health concern. Cell entry of SARS-CoV-2 depends on viral spike (S) proteins binding to cellular receptors (ACE2) and their subsequent priming by host cell proteases (TMPRSS2). Assessing effects of viral-induced host response factors and determining which cells are used by SARS-CoV-2 for entry might provide insights into viral transmission, add clarity to the virus' pathogenesis, and possibly reveal therapeutic targets. Mast cells (MCs) are ubiquitously expressed tissue cells that act as immune sentinels given their ability to react specifically to pathogens at environmental interfaces, such as in the lung. Several lines of evidence suggest a critical role for MCs in SARS-CoV-2 infections based on patients' mediator profiles, especially the "cytokine storm" responsible for most morbidity and mortality. In this pilot study, we demonstrated that human lung MCs (n = 3 donors) are a source of renin and that they upregulate the membrane receptor for SARS-CoV-2 (ACE2) as well as the protease required for cellular entry (TMPRSS2) under certain conditions. We hypothesized that infection of human MCs with SARS-CoV-2 may be a heretofore-unrecognized mechanism of viral pathogenesis, and further studies are required to assess this question.

Description and Characterization of Three-Dimensional Human Mast Cell Progenitor Spheroids In Vitro.

Human mast cells (MC) are an essential component of the immune system as they uniquely store and release a wide range of soluble mediators through IgE and non-IgE mechanisms. Several tissue sources can be used to differentiate functional MC for in vitro and in vivo studies. Here we describe an improved method for obtaining large numbers of human MC from adipose tissue with advantages over current methods. We analyzed donor parameters (e.g. age, race) on MC-isolation following adipose and skin tissue digestion from healthy donors. Adipose and skin-derived MC were morphologically and immunophenotypically similar in all donors regardless of age. However, donor-dependent variations in MC numbers were observed following tissue digestion. In addition, we identified and characterized three-dimensional structures from which mature MC emerged in vitro using peripheral blood and human tissue sources. MC progenitor spheroids (MCPS) appeared approximately one week following progenitor isolation and were consistently observed to have mature MC attached, emerging, or nearby when cultured in a stem cell factor-containing medium. The overall characteristics of the MCPS were similar from each tissue source. We propose that these MCPS serve as the common source of human MC in vitro.

Fullerene antioxidants decrease organophosphate-induced acetylcholinesterase inhibition in vitro.

Although organophosphate (OP)-induced acetylcholinesterase (AChE) inhibition is the critical mechanism causing toxicities that follow exposure, other biochemical events, including oxidative stress, have been reported to contribute to OP toxicity. Fullerenes are carbon spheres with antioxidant activity. Thus, we hypothesized that fullerenes could counteract the effects of OP compounds and tested this hypothesis using two in vitro test systems, hen brain and human neuroblastoma SH-SY5Y cells. Cells were incubated with eight different derivatized fullerene compounds before challenge with paraoxon (0=control, 5×10(-8), 10(-7), 2×10(-7) or 5×10(-7) M) or diisopropylphosphorofluoridate (DFP, 0=control, 5×10(-6), 10(-5), 2×10(-5), and 5×10(-5) M) and measurement of AChE activities. Activities of brain and SH-SY5Y AChE with OP compounds alone ranged from 55-83% lower than non-treated controls after paraoxon and from 60-92% lower than non-treated controls after DFP. Most incubations containing 1 and 10 µM fullerene derivatives brought AChE activity closer to untreated controls, with improvements in AChE activity often >20%. Using dissipation of superoxide anion radicals as an indicator (xanthine oxidation as a positive control), all fullerene derivatives demonstrated significant antioxidant capability in neuroblastoma cells at 1 µM concentrations. No fullerene derivative at 1 µM significantly affected neuroblastoma cell viability, when determined using either Alamar Blue dye retention or a luminescent assay for ATP production. These studies suggest that derivatized fullerene nanomaterials have potential capability to ameliorate OP-induced AChE inhibition resulting in toxicities.

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation.

OBJECTIVE: Mast cells are tissue-resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis. METHODS: In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR(-/-) and control mast cells were engrafted into mast cell-deficient WBB6 F1-Kit(w) /Kit(W-v) (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR(-/-) animals and in murine and human mast cell cultures. RESULTS: Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR(-/-) mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR(-/-) mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin-8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR(-/-) mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis. CONCLUSION: Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a-modulated expression of FcγR.

"Accentuate the negative, eliminate the positive": engineering allergy therapeutics to block allergic reactivity through negative signaling.

By targeting the dominant-negative signaling receptor FcgammaRIIb expressed on proallergic cells, we have developed 2 novel platforms for the treatment of IgE-mediated allergic disease. First is a genetically engineered bifunctional human fusion protein GE2, which is comprised of the Fc portions of human IgE and IgG1 with an interposed flexible linker designed as a long-term parenteral allergen-nonspecific therapy. GE2 blocks the effector phase of the IgE response in vitro in mice and human subjects and in vivo in the skin and airway and systemically in mice and monkeys. Whether reactivity against human GE2 in human subjects will limit its applicability remains to be determined. The second platform is designed to provide a safer form of allergen-specific immunotherapy and consists of genetically engineered chimeric human Fcgamma-allergen proteins, with Fcgamma-Fel d 1 as the prototype. The allergen portion binds to specific IgE on FcepsilonRs, whereas the Fcgamma portion coaggregates inhibitory FcgammaRIIb and drives inhibition of allergic reactivity. Fcgamma-Fel d 1 blocked human mast cell Fel d 1-induced allergic reactivity in vitro and in vivo in murine models while functioning as an immunogen but not as an allergen.

IL-4 selectively enhances FcgammaRIII expression and signaling on mouse mast cells.

Fc receptors for IgG (FcgammaR) are widely expressed in the hematopoietic system and mediate a variety of inflammatory responses. There are two functional classes of FcgammaR, activation and inhibitory receptors. Since IgG immune complexes (IgG IC) bind each class with similar affinity, co-expression of these receptors leads to their co-ligation. Thus, expression levels of this antagonistic pair play a critical role in determining the cellular response. Murine mast cells co-express the activation receptor FcgammaRIII and the inhibitory receptor FcgammaRIIb and can be activated by IgG IC. Mast cell activation contributes to allergic and other inflammatory diseases-particularly those in which IgG IC may play important roles. Using mouse bone marrow-derived mast cells, we report that IL-4 selectively increases FcgammaRIII expression without altering FcgammaRIIb. This enhanced expression could be induced by Stat6 activation alone, and appeared to be mediated in part by increased FcgammaRIIIalpha protein synthesis without significant changes in transcription. The increase in FcgammaRIII expression was functionally significant, as it was matched by enhanced FcgammaR-mediated degranulation and cytokine production. Selective regulation of mast cell FcgammaR by interleukin-4 could alter inflammatory IgG responses and subsequently disease severity and progression.