Th17 Turmoil in Metabolic Dysfunction See article p.3287

Foiling Phagocytosis To Ameliorate Inflammation See article p.3331

Giving ADIN a TRIM See article p.3452

Rac-ing Up Isoform Dynamics See article p.3479

Antibody targeting of the adhesion molecule CD44 has been used in animal models for the treatment of inflammatory and autoimmune diseases, some of which may involve dysregulation of phagocytosis. Phagocytosis is induced by a variety of surface receptors, including Fcγ receptors (FcγRs), complement receptors, and nonopsonic receptors such as dectin-1. In this issue, Amash et al. (p. 3331) investigate the influence of the well-studied anti-CD44 mAb IM7 on macrophage phagocytosis. IM7 pretreatment of RAW264.7 (RAW) macrophages or primary peritoneal macrophages prevented FcγR-mediated phagocytosis of IgG-opsonized SRBCs in a dose-dependent manner; phagocytic inhibition was lower in primary macrophages than in RAW cells, potentially due to population heterogeneity. Other anti-CD44 mAbs investigated were unable to inhibit FcγR-mediated phagocytosis of RAW macrophages at any dose tested. IM7 could inhibit macrophage binding to target cells and was also able to prevent phagocytosis when added after macrophage-target cell binding, suggesting that IM7 interferes with phagocytosis at multiple stages. Although the F(ab′)2 form of IM7 was capable of binding RAW cells, it could not inhibit phagocytosis, implicating the Fc portion in this process. IM7 also inhibited complement receptor 3–mediated phagocytosis of C3bi-opsonized SRBCs, but not dectin-1-mediated phagocytosis of zymosan particles. Collectively, these results show that the anti-CD44 mAb IM7 enacts some of its potent anti-inflammatory effects by inhibiting opsonic receptor-mediated phagocytosis.

Obesity increases the risks of cancer and autoimmunity, but a fraction of obese individuals do not have metabolic complications and are termed metabolically healthy obese (MHO), in contrast to metabolically unhealthy obese (MUO) individuals. Local inflammation in non-lymphoid tissues such as visceral adipose tissue (VAT) is influenced by tissue-resident T cells, including regulatory T cells (Tregs) and polarized effector T cells such as Th17 cells. The ectonuclease CD39 mediates the first step in ATP conversion and is able to inhibit ATP-induced apoptosis. Stress signals, including extracellular ATP, can travel through the purinergic P2X7 receptor (P2X7R) and drive Th17 inflammatory responses, prompting Pandolfi et al. (p. 3287) to investigate the role that P2X7R plays in driving Th17 responses in obesity. Human VAT explants cultured from MUO donors secreted significantly more IL-6, IL-1β, IL-17, and IL-23 than explants from either lean or MHO donors. Treatment with the P2X7R antagonist KN-62 reduced production of these inflammatory cytokines only in explants from MUO donors, indicating that tissue purinergic signaling could promote a Th17 polarizing environment in MUO individuals. Expression of the Th17 master transcription factor RORC was significantly higher in MUO stromal vascular cells than in MHO or lean cells, and there was also a higher proportion of IL-17+ CD4+ and IL-23R+ CD4+ T cells from MUO donors. CD39, normally associated with Tregs, was coexpressed with IL-23R on CD4+ T cells from MUO donors more frequently than on cells from MHO or lean donors; however, these cells were unlikely to be Tregs, as VAT cells from MUO donors had more CD39+Foxp3- CD4+ T cells relative to lean or MHO donor samples. Interestingly, the metabolic score of donors positively correlated with the proportion of CD39+Foxp3- CD4+ and IL-23R+ CD4+ T cells, as well as with levels of RORC expression. CD39+ CD4+ T cells from lean donors were more resistant to apoptosis following treatment with a P2X7R agonist than were CD39-Foxp3+ CD4+ T cells. These results indicate that ATP-induced apoptosis through P2X7R signaling may differentially affect T cell subsets in VAT, leading to Th17 skewing in MUO individuals.

Antibodies bound to viruses can be brought via Fcγ receptors (FcγRs) into the cell, where they can interact with cytosolic immune receptors such as tripartite motif containing-21 (TRIM21), which activates NF-κB signaling and initiates Ab-dependent intracellular neutralization (ADIN). This process is mediated via TRIM21 binding to the Fc portion of the Ab and targeting the Ab-virus complex for proteasomal degradation, releasing the viral genome and activating cytosolic nucleic acid sensors such as RIG-I and cGAS. TRIM21 binds IgG with high affinity, and Foss et al. (p. 3452) examined how this binding affinity affects downstream immune signaling. The authors generated a humanized IgG1 mAb, h9C12, which bound with its target human adenovirus 5 (AdV5)-GFP and induced ADIN and activated NF-κB in human cell lines. Using information from the crystal structure of the IgG1 Fc with the binding domain of human TRIM21, the authors generated a panel of h9C12 mutants to test the differential effect of TRIM21-IgG1 Fc binding affinity on ADIN and NF-κB activation. Although none of the mutations destabilized the IgG molecule, surface plasmon resonance with the TRIM21 monomeric binding domain and ELISA with full-length TRIM21 showed the mutations resulted in changes in affinity ranging from a 2-fold to over a 100-fold reduction. Some Ab mutations that eliminated binding to FcγR and C1q did not affect TRIM21 binding, ADIN, or NF-κB activation. Overall, NF-κB activation was more sensitive than ADIN to TRIM21 binding affinity reduction. Low levels of opsonization also had more of an impact on NF-κB activation than ADIN, indicating that ADIN may occur even with low Ab levels. These observations have important implications for the design of engineered IgG antiviral therapeutics.

The p21 Rho family is made up of small GTPases with four major isoforms (Rac1, Rac2, Rac3, and RhoG). Although both Rac1 and Rac2 are expressed in immune cells, they have variable expression levels and non-redundant functions in many actin-dependent leukocyte processes, including development, chemotaxis, phagocytosis, and ROS production. Subcellular localization of Rac1 and Rac2 is thought to play an important role in their functional non-redundancy; however, limitations in current visualization techniques make simultaneous analysis of these two isoforms cumbersome. To dissect the disparate roles of Rac1 and Rac2 in macrophage activation, Miskolci et al. (p. 3479) created a Rac2 genetically encoded single-chain Förster resonance energy transfer (FRET)-based biosensor. The authors also optimized previously engineered genetically encoded Rac1 and Cdc42 biosensors to allow the direct concurrent visualization of several Rho GTPases in macrophage cell lines. These genetically encoded biosensors improve upon biomolecular biosensors by maintaining the integrity of the C-terminal polybasic tail, allowing for correct intracellular localization and interaction with upstream regulators, and eliminating the need to correct for nonequimolar distribution of FRET donor/acceptor components during analyses. With the use of these biosensors in macrophage cell lines, the authors found that Rac1 and Rac2 had similar activation kinetics but disparate spatial distribution in actin-rich protrusions elicited in response to formyl-methionyl-leucyl-phenylalanine stimulation. Rac1 distributed to the cell edges, whereas Rac2 had broader distribution throughout the cell with a concentration of activity in the perinuclear region of the cell body. Rac2 was activated first, distal to the protrusion, followed by Rac1, a secondary Rac2 activation, and Cdc42 activations behind the leading edge of initiated protrusion. Together, these results suggest that isoform-specific biosensors optimized for expression will be useful in deciphering complex intracellular dynamics of proteins within living cells.