Mitochondrial dysfunction (MD) and inflammaging are hallmarks of non-communicable diseases and play a pivotal role in the ageing process. Determination of mitochondrial function (MF) in Peripheral Blood Mononuclear Cells (PBMCs) represents a minimally invasive method for assessing the pathophysiological state. However, the necessity to measure the cells in a fresh state is a challenge especially in multicenter investigational studies. Therefore, we investigated the mitochondrial and immunological properties of cryopreserved PBMC isolated from whole blood and buffy coats over a storage period of 6 months to establish a procedure to assess MD in frozen samples.
PBMCs were isolated from whole blood and buffy coats using gradient density centrifugation and cryopreserved with fetal bovine serum and dimethyl sulfoxide as antifreeze agents in liquid nitrogen. To identify potential differences between cryopreserved and freshly isolated cells, we investigated cell count and viability, mitochondrial respiration, adenosine triphosphate production, and citrate synthase activity in PBMCs during 6 months of storing. Cell composition was determined using flow cytometry. The immune status was assessed by determining the cytokines IL-6, IL-10, and TNF-α after phytohemagglutinin stimulation using time-resolved fluorescence (HTRF).
After cryopreservation and storage of PBMCs isolated from whole blood or buffy coats, their bioenergetic function is preserved for at least 6 months: No statistically significant differences in the parameters CS, ATP, and the complexes of the respiratory chain were observed at any measurement time point. However, fluorescence-activated cell sorting analysis reveals that the number of apoptotic cells increased after 1 month of cryopreservation. After 3 months of cryopreservation, phytohemagglutinin-activated cytokines IL-6, IL-10 and TNF-α were significantly increased, indicating a more sensitized immune response of frozen cells.
Cryopreservation of PBMCs has no effect on the measurement of bioenergetic parameters, although increased apoptotic cells are measured. Cryopreserved PBMCs show an increased immunological response, which must be taken into account when interpreting the results.
Copyright © 2026 Dieter, Grube, Quentin and Eckert.

Multiple p38 MAP kinase inhibitors have been developed for the treatment of inflammatory diseases such as rheumatoid arthritis, but their effectiveness has been limited due to toxicity and tachyphylaxis, leading to a lack of clinical benefit. Efforts have been made to circumvent this limitation by targeting individual substrates downstream of p38, including MK2 and MK5. This approach has failed to yield clinical benefit despite preclinical evidence of a therapeutic effect. We hypothesized that there is redundancy in the MAPK activating kinase family that would necessitate blocking multiple kinases to sufficiently impact inflammatory processes. We used heterobifunctional protein degraders that either specifically degraded MK2 selectively or degraded MK2/3/5 simultaneously to test the hypothesis, in addition to genetic approaches to enable knockdown. In human PBMCs, elimination of MK2/3/5 with heterobifunctional degraders resulted in full reduction of TLR4 or TLR7/8 induced TNFα, whereas MK2-specific degradation only attenuated TNFα biosynthesis. In contrast, both specific MK2 degradation and broad MK2/3/5 degradation inhibited TGF-β-induced collagen production in human fibroblasts. This observation was consistent with genetic deletions of MK2, MK3 and MK5 (singly and in combination) whereby single deletion of MK2, MK3 or MK5 attenuated lipopolysaccharide (LPS) induced TNFα production and had no effect on R848-induced TNFα production. Double deletion of MK2 and MK3 or MK2 and MK5 or MK2/3/5 triple deletion had a significantly greater effect on TNFα production regardless of stimulus. The combined data suggest cooperativity between MK2 and either MK3 or MK5 for efficient, cell context-dependent modulation of inflammatory responses.
Copyright © 2026 Yang, Fang, Marx, Liu, Skouras, Sharma, Walther, Bollinger Martinez, Hubeau, Shi, De Savi, Huang, Huhn, Sawant, Proctor, Dixit, Dong, Weiss, Mainolfi, Slavin, Long, Williams and Byrne.

Bruton's tyrosine kinase (BTK) is an intracellular signaling enzyme that regulates B-lymphocyte and myeloid cell functions. Due to its involvement in both innate and adaptive immune compartments, BTK inhibitors have emerged as a therapeutic option in autoimmune disorders such as multiple sclerosis (MS). Brain-penetrant, small-molecule BTK inhibitors may also address compartmentalized neuroinflammation, which is proposed to underlie MS disease progression. BTK is expressed by microglia, which are the resident innate immune cells of the brain; however, the precise roles of microglial BTK and impact of BTK inhibitors on microglial functions are still being elucidated. Research on the effects of BTK inhibitors has been limited to rodent disease models. This is the first study reporting effects in human microglia.
Here we characterize the pharmacological and functional properties of fenebrutinib, a potent, highly selective, noncovalent, reversible, brain-penetrant BTK inhibitor, in human microglia and complex human brain cell systems, including brain organoids.
We find that fenebrutinib blocks the deleterious effects of microglial Fc gamma receptor (FcγR) activation, including cytokine and chemokine release, microglial clustering and neurite damage in diverse human brain cell systems. Gene expression analyses identified pathways linked to inflammation, matrix metalloproteinase production and cholesterol metabolism that were modulated by fenebrutinib treatment. In contrast, fenebrutinib had no significant impact on human microglial pathways linked to Toll-like receptor 4 (TLR4) and NACHT, LRR and PYD domains-containing protein 3 (NLRP3) signaling or myelin phagocytosis.
Our study enhances the understanding of BTK functions in human microglial signaling that are relevant to MS pathogenesis and suggests that fenebrutinib could attenuate detrimental microglial activity associated with FcγR activation in people with MS.
© 2024. The Author(s).

Mucopolysaccharidoses are inherited metabolic disorders caused by the deficiency in lysosomal enzymes required to break down glycosaminoglycans. Accumulation of glycosaminoglycans leads to progressive, systemic degenerative disease. The central nervous system is particularly affected, resulting in developmental delays, neurological regression, and early mortality. Current treatments fail to adequately address neurological defects. Here we explore the potential of human induced pluripotent stem cell (hiPSC)-derived microglia progenitors as a one-time, allogeneic off-the-shelf cell therapy for several mucopolysaccharidoses (MPS). We show that hiPSC-derived microglia progenitors, possessing normal levels of lysosomal enzymes, can deliver functional enzymes into four subtypes of MPS knockout cell lines through mannose-6-phosphate receptor-mediated endocytosis in vitro. Additionally, our findings indicate that a single administration of hiPSC-derived microglia progenitors can reduce toxic glycosaminoglycan accumulation and prevent behavioral deficits in two different animal models of MPS. Durable efficacy is observed for eight months after transplantation. These results suggest a potential avenue for treating MPS with hiPSC-derived microglia progenitors.
© 2024. The Author(s).

G protein-coupled receptors (GPCRs) are key regulatory proteins of immune cell function inducing signaling in response to extracellular (pathogenic) stimuli. Although unrelated, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 share signaling via Gαi/o proteins and the agonist 3-hydroxydecanoic acid (3HDec). Both receptors are abundantly expressed in monocytes, macrophages and neutrophils but have opposing functions in these innate immune cells. Detailed insights into the molecular mechanisms and signaling components involved in immune cell regulation by GPR84 and HCA3 are still lacking. Here, we report that GPR84-mediated pro-inflammatory signaling depends on coupling to the hematopoietic cell-specific Gα15 protein in human macrophages, while HCA3 exclusively couples to Gαi protein. We show that activated GPR84 induces Gα15-dependent ERK activation, increases intracellular Ca2+ and IP3 levels as well as ROS production. In contrast, HCA3 activation shifts macrophage metabolism to a less glycolytic phenotype, which is associated with anti-inflammatory responses. This is supported by an increased release of anti-inflammatory IL-10 and a decreased secretion of pro-inflammatory IL-1β. In primary human neutrophils, stimulation with HCA3 agonists counteracts the GPR84-induced neutrophil activation. Our analyses reveal that 3HDec acts solely through GPR84 but not HCA3 activation in macrophages. In summary, this study shows that HCA3 mediates hyporesponsiveness in response to metabolites derived from dietary lactic acid bacteria and uncovers that GPR84, which is already targeted in clinical trials, promotes pro-inflammatory signaling via Gα15 protein in macrophages.
Copyright © 2021 Elsevier Ltd. All rights reserved.