Manipulating immune activity of macrophages: a materials and mechanics perspective.

Macrophage immune cells exist on a plastic spectrum of phenotypes governed by their physical and biochemical environment. Controlling macrophage function to facilitate immunological regeneration or fighting pathology has emerged as a therapeutic possibility. The rate-limiting step in translating macrophage immunomodulation therapies has been the absence of fundamental knowledge of how physics and biochemistry in the macrophage microenvironment converge to inform phenotype. In this review we explore recent trends in bioengineered model systems that integrate physical and biochemical variables applied to macrophage mechanosensing and plasticity. We focus on how tuning of mechanical forces and biomaterial composition orchestrate macrophage function in physiological and pathological contexts. Ultimately, a broader understanding of stimuli-responsiveness in macrophages leads to informed design for future modulatory therapies.

Site-Specific Glycosylation Analysis of Human and Murine Fcγ Receptor II Family Members Reveals Variant-Specific N-Glycosylation.

Fcγ-receptors (FcγRs) including FcγRII (CD32) gene family members are expressed on leukocytes, bind the crystallizable fragment (Fc) region of immunoglobulin G (IgG), and bridge humoral and cellular immunity. FcγRIIA and FcγRIIB have opposing roles, with the former responsible for activation and the latter for inhibition of immune cell signaling and effector functions. The extracellular domains of human and murine FcγRIIs share multiple conserved N-glycosylation sites. Understanding the role(s) of FcγRIIA and FcγRIIB glycosylation in autoimmune diseases is precluded by a lack of effective methods to study disease-associated changes in glycosylation. To address this barrier, we developed a method to assess site-specific glycosylation of human FcγRIIA and FcγRIIB, and the mouse ortholog of human FcγRIIB. Among the receptors, conserved glycosylation sites are compared, with the N144/145 site displaying predominantly complex glycans in recombinant FcγRIIs. Differences in sialylation between recombinant human FcγRIIA H/R134 (H/R131) variants at a nearby N145 N-glycosylation site are reported. Further, a potential human FcγRIIA O-glycosylation site, S179 (S212), is reported in recombinant FcγRIIA. The robust method to assess site-specific glycosylation of FcγRIIs reported here, can be utilized to study the potential role of FcγRII family glycosylation in disease. Data are available via ProteomeXchange with identifier PXD049429.

Clonal Dynamics and Relapse Risk Revealed by High-Sensitivity FLT3-Internal Tandem Duplication Detection in Acute Myeloid Leukemia.

The ability to detect low-level disease is key to our understanding of clonal heterogeneity in acute myeloid leukemia (AML) and residual disease that elude conventional assays and seed relapse. We developed a high-sensitivity next-generation sequencing (HS-NGS) clinical assay, able to reliably detect low levels (1 × 10-5) of FLT3-ITD, a frequent, therapeutically targetable and prognostically relevant mutation in AML. By applying this assay to 289 longitudinal samples from 62 patients at initial diagnosis and/or clinical follow-up (mean follow-up of 22 months), we reveal the frequent occurrence of FLT3-ITD subclones at diagnosis and demonstrate a significantly decreased relapse risk when FLT3-ITD is cleared after induction or thereafter. We perform pairwise sequencing of diagnosis and relapse samples from 23 patients to uncover more detailed patterns of FLT3-ITD clonal evolution at relapse than is detectable by less-sensitive assays. Finally, we show that rising ITD level during consecutive biopsies is a harbinger of impending relapse. Our findings corroborate the emerging clinical utility of high-sensitivity FLT3-ITD testing and expands our understanding of clonal dynamics in FLT3-ITD-positive AML.

Introduction to Biomaterials in Innate Immunity.

Erika Moore and Shreya A Raghavan introduce the Journal of Materials Chemistry B and Materials Advances joint themed issue on Biomaterials in Innate Immunity.

Metformin Treatment of Macrophages Increases Microvessel Growth in Three-Dimensional Hydrogel Coculture.

The global population is aging rapidly, posing unprecedented challenges to health care systems. This study investigates the often-overlooked role of macrophages in microvascular dysfunction associated with aging. We use a three-dimensional in vitro hydrogel model to assess the effects of both age and metformin, an anti-aging therapeutic, on macrophage interactions with microvasculature. Metformin's broad cellular impact is a subject of significant interest, yet its precise mechanisms remain unclear. Our research reveals that metformin treatment enhances genetic pathways associated with macrophage-mediated support of angiogenesis, resulting in increased microvessel density. Of importance, monocyte chemoattractant protein-1 expression is upregulated with metformin treatment and positively correlated with microvascular volume, shedding light on a potential mechanism for metformin's promotion of macrophage support of vasculogenesis. This work not only uncovers metformin's impact on human macrophages but also supports its potential as an antiaging therapeutic, offering new avenues for combating age-related diseases.

Laminin-derived peptide, IKVAV, modulates macrophage phenotype through integrin mediation.

Macrophages are highly plastic immune cells known to exist on a spectrum of phenotypes including pro-inflammatory (M1) or pro-healing (M2). Macrophages interact with extracellular matrix (ECM) ligands, such as fragments of collagen and laminin. Interaction of macrophages with ECM ligands is mediated through integrin receptors. However, the role of ECM ligands in directing macrophage function through integrins is not yet fully understood. Particularly, α2ß1 has been implicated in modulating macrophage function, but complexity in mechanisms employed for integrin-ligation especially with laminin-derived peptides makes it challenging to understand macrophage-ECM interactions. We hypothesize that targeting α2ß1 through laminin-derived peptide, IKVAV, will modulate macrophage phenotype. In this work we: i) investigated macrophage response to IKVAV in 2D and in a 3D platform, and ii) identified α2ß1's role as it pertains to macrophage modulation via IKVAV. Soluble IKVAV treatment significantly reduced M1 markers and increased M2 markers via immunocytochemistry and gene expression. While the 3D ECM-mimicking PEG-IKVAV hydrogels did not have significant effects in modulating macrophage phenotype, we found that macrophage modulation via IKVAV is dependent on the concentration of peptide used and duration of exposure. To investigate integrin-ligand interactions for macrophages, α2ß1 signaling was modulated by antagonists and agonists. We observed that blocking α2ß1 reduces M1 activation. To understand integrin-ligand interactions and leveraging the therapeutic ability of macrophages in designing immunomodulatory solutions, it is critical to elucidate IKVAV's role in mediating macrophage phenotype.

Site-Specific Glycosylation Analysis of Murine and Human Fcγ Receptors Reveals High Heterogeneity at Conserved N-Glycosylation Site.

Fc γ-receptors (FcγRs) on leukocytes bind immunoglobulin G (IgG) immune complexes to mediate effector functions. Dysregulation of FcγR-mediated processes contributes to multiple inflammatory diseases, including rheumatoid arthritis, lupus, and immune thrombocytopenia. Critically, immunoregulatory N-glycan modifications on both FcγRs and IgGs alter FcγR-IgG binding affinity. Rapid methods for the characterization of N-glycans across multiple Fcγ receptors are needed to propel investigations into disease-specific contributions of FcγR N-glycans. Here, we utilize nanoliquid chromatography tandem mass spectrometry (nLC-MS/MS) to characterize FcγR glycosylation and report quantitative and site-specific N-glycan characterization of recombinant human FcγRI, FcγRIIIA V158, and FcγRIIIA F158 from CHO cells and murine FcγRI, FcγRIII, and FcγRIV from NS0 cells. Data are available via ProteomeXchange with identifier PXD043966. Broad glycoform distribution (≥30) was observed at mouse FcγRIV site N159 and human FcγRIIIA site N162, an evolutionarily conserved site. Further, mouse FcγRIII N-glycopeptides spanning all four predicted N-glycosylation sequons were detected. Glycoform relative abundances for hFcγRIIIA V/F158 polymorphic variants are reported, demonstrating the clinical potential of this workflow to measure differences in glycosylation between common human FcγRIIIA allelic variants with disease-associated outcomes. The multi-Fcγ receptor glycoproteomic workflow reported here will empower studies focused on the role of FcγR N-glycosylation in autoimmune diseases.

IGJ and SPATS2L immunohistochemistry sensitively and specifically identify BCR::ABL1+ and BCR::ABL1-like B-acute lymphoblastic leukaemia.

Therapeutic management and prognostication for patients with B-acute lymphoblastic leukaemia (B-ALL) require appropriate disease subclassification. BCR::ABL1-like B-ALL is unique in that it is defined by a gene expression profile similar to BCR::ABL1+ B-ALL rather than a unifying recurrent translocation. Current molecular/cytogenetic techniques to identify this subtype are expensive, not widely accessible, have long turnaround times and/or require an adequate liquid biopsy. We have studied a total of 118 B-ALL cases from three institutions in two laboratories to identify surrogates for BCR::ABL1+/like B-ALL. We report that immunoglobulin joining chain (IGJ) and spermatogenesis associated serine-rich 2-like (SPATS2L) immunohistochemistry (IHC) sensitively and specifically identify BCR::ABL1+/like B-ALL. IGJ IHC positivity has a sensitivity of 83%, a specificity of 95%, a positive predictive value (PPV) of 89% and a negative predictive value (NPV) of 90%. SPATS2L staining has similar sensitivity and NPV but lower specificity (85%) and PPV (70%). The presence of either IGJ or SPATS2L staining augments the sensitivity (93%) and NPV (95%). While these findings would need to be validated in larger studies, they suggest that IGJ and/or SPATS2L IHC may be utilized in identifying BCR::ABL1-like B-ALL or in selecting B-ALL cases for confirmatory molecular/genetic testing, particularly in resource-limited settings.

Age-associated Senescent - T Cell Signaling Promotes Type 3 Immunity that Inhibits the Biomaterial Regenerative Response.

Aging is associated with immunological changes that compromise response to infections and vaccines, exacerbate inflammatory diseases and can potentially mitigate tissue repair. Even so, age-related changes to the immune response to tissue damage and regenerative medicine therapies remain unknown. Here, it is characterized how aging induces changes in immunological signatures that inhibit tissue repair and therapeutic response to a clinical regenerative biological scaffold derived from extracellular matrix. Signatures of inflammation and interleukin (IL)-17 signaling increased with injury and treatment both locally and regionally in aged animals, and computational analysis uncovered age-associated senescent-T cell communication that promotes type 3 immunity in T cells. Local inhibition of type 3 immune activation using IL17-neutralizing antibodies improves healing and restores therapeutic response to the regenerative biomaterial, promoting muscle repair in older animals. These results provide insights into tissue immune dysregulation that occurs with aging that can be targeted to rejuvenate repair.

The clinical significance of increased large cells in marginal zone lymphoma.

Marginal zone lymphoma (MZL) is a primary, indolent small B-cell lymphoma. Subtypes include nodal, splenic, and those of extranodal mucosa-associated lymphoid tissue (MALT). These are slow growing and generally exhibit low rates of transformation to diffuse large B-cell lymphoma (DLBCL). At initial diagnosis, there can be an increase in large cells (LCs) that does not meet criteria for DLBCL. Prior studies have noted this finding, but the clinical significance of these LCs has not been well established. A total of 161 cases of MZL from 1994 to 2021 were evaluated, including all subtypes. There were 33 cases with increased LCs (>10 LCs per high-power field [hpf]), with the majority containing >15 LCs/hpf (28/33) and 128 cases without increased LCs. Cases with increased LCs were significantly more likely to have a Ki-67 proliferation index of ≥30% (P < .0001). Overall survival was not significantly different between the groups but progression-free survival was significantly worse in the LC group (P < .0001). MZL with increased LCs was also associated with a higher stage at diagnosis (P = .0035), was more likely to transform to DLBCL (P = .0016), and had a greater frequency of relapse (P < .0001). Subgroup analysis showed that both nodal and MALT LC groups had a worse progression-free survival and a higher rate of relapse than their standard nodal and MALT lymphoma counterparts, but only within the MALT subgroup did the LC cases present at a higher stage and have a higher rate of transformation to DLBCL than the standard cases. Although larger studies are needed for validation, these results suggest that the presence of LCs in MZL may serve as a useful prognostic indicator and potentially help guide clinical decision-making.

Generation and characterization of two immortalized dermal fibroblast cell lines from the spiny mouse (Acomys).

The spiny mouse (Acomys) is gaining popularity as a research organism due to its phenomenal regenerative capabilities. Acomys recovers from injuries to several organs without fibrosis. For example, Acomys heals full thickness skin injuries with rapid re-epithelialization of the wound and regeneration of hair follicles, sebaceous glands, erector pili muscles, adipocytes, and dermis without scarring. Understanding mechanisms of Acomys regeneration may uncover potential therapeutics for wound healing in humans. However, access to Acomys colonies is limited and primary fibroblasts can only be maintained in culture for a limited time. To address these obstacles, we generated immortalized Acomys dermal fibroblast cell lines using two methods: transfection with the SV40 large T antigen and spontaneous immortalization. The two cell lines (AcoSV40 and AcoSI-1) maintained the morphological and functional characteristics of primary Acomys fibroblasts, including maintenance of key fibroblast markers and ECM deposition. The availability of these cells will lower the barrier to working with Acomys as a model research organism, increasing the pace at which new discoveries to promote regeneration in humans can be made.

SOCS1-KIR Peptide in PEGDA Hydrogels Reduces Pro-Inflammatory Macrophage Activation.

Macrophages modulate the wound healing cascade by adopting different phenotypes such as pro-inflammatory (M1) or pro-wound healing (M2). To reduce M1 activation, the JAK/STAT pathway can be targeted by using suppressors of cytokine signaling (SOCS1) proteins. Recently a peptide mimicking the kinase inhibitory region (KIR) of SOCS1 has been utilized to manipulate the adaptive immune response. However, the utilization of SOCS1-KIR to reduce pro-inflammatory phenotype in macrophages is yet to be investigated in a biomaterial formulation. This study introduces a PEGDA hydrogel platform to investigate SOCS1-KIR as a macrophage phenotype manipulating peptide. Immunocytochemistry, cytokine secretion assays, and gene expression analysis for pro-inflammatory macrophage markers in 2D and 3D experiments demonstrate a reduction in M1 activation due to SOCS1-KIR treatment. The retention of SOCS1-KIR in the hydrogel through release assays and diffusion tests is demonstrated. The swelling ratio of the hydrogel also remains unaffected with the entrapment of SOCS1-KIR. This study elucidates how SOCS1-KIR peptide in PEGDA hydrogels can be utilized as an effective therapeutic for macrophage manipulation.

What does Juneteenth mean in STEMM?

Juneteenth commemorates the freeing of the last large group of enslaved people in 1865 at the end of the American Civil War. We asked several Black scientists what Juneteenth means to them in the context of science, technology, engineering, mathematics, and medicine (STEMM)? Their answers run the emotional gamut.

Helminth egg derivatives as proregenerative immunotherapies.

The immune system is increasingly recognized as an important regulator of tissue repair. We developed a regenerative immunotherapy from the helminth Schistosoma mansoni soluble egg antigen (SEA) to stimulate production of interleukin (IL)-4 and other type 2-associated cytokines without negative infection-related sequelae. The regenerative SEA (rSEA) applied to a murine muscle injury induced accumulation of IL-4-expressing T helper cells, eosinophils, and regulatory T cells and decreased expression of IL-17A in gamma delta (γδ) T cells, resulting in improved repair and decreased fibrosis. Encapsulation and controlled release of rSEA in a hydrogel further enhanced type 2 immunity and larger volumes of tissue repair. The broad regenerative capacity of rSEA was validated in articular joint and corneal injury models. These results introduce a regenerative immunotherapy approach using natural helminth derivatives.

A Biomaterial Model to Assess the Effects of Age in Vascularization.

As humans age, there is an increased risk for developing age-associated diseases. Many of these diseases, such as cardiovascular disease, involve dysfunction in the vasculature. Cardiovascular disease stems from endothelial cell dysfunction and reduction in vascularization. Macrophages, prominent innate immune cells involved in orchestrating inflammation and wound healing, have a significant influence on vascularization. While much recent work has investigated the crosstalk between endothelial cells and macrophages, it is still not well defined. The interactions between the cell types are even less understood in specific disease states such as advanced age. Understanding how age influences macrophage/endothelial cell interaction is essential for understanding cardiovascular disease development in the elderly. In the polyethylene glycol (PEG)-based hydrogel system, we model the effects of age on vascularization by encapsulating endothelial cells, pericytes, and human donor macrophages. We created a biomaterial model system in which macrophages, either from young (<35 years old) or old (>65 years old) donors, interact with the modeled vasculature, termed microvessels. Confocal image analysis of vessel density, vessel length, and branch points were used to quantify microvessel growth depending on the age of the macrophage donor. Alongside this, soluble factor secretion and gene expression were evaluated using ELISA and NanoString to showcase biological mechanisms based on the age of each donor. Endothelial cells cultured with macrophages from old donors have reduced microvessel density. There also is reduced soluble factor secretion by the macrophages from old donors, which likely influenced microvessel growth. Altogether, we establish our PEG-based hydrogel vascular model as a system to evaluate patient-specific cell function as well as proposed mechanisms for how age influences microvessels.

An in vitro study of micromechanics, cellular proliferation and viability on both decellularized porcine dura grafts and native porcine dura grafts.

Damage to the dura mater may occur during intracranial or spinal surgeries, which can result in cerebrospinal fluid leakage and other potentially fatal physiological changes. As a result, biological and synthetic derived scaffolds are typically used to repair dura mater post intracranial or spinal surgeries. The extracellular matrix of xenogeneic dura scaffolds has been shown to exhibit increased cell infiltration and regeneration than synthetic dura materials. In this study, we investigated the biocompatibility of native and decellularized porcine dura by seeding rat fibroblast cells onto the constructs. Cell proliferation, cell viability, and the mechanical properties of these dural grafts were evaluated post-re-seeding on days 3,7 and 14. Live-dead staining and resazurin salts were used to quantify cell viability and cell proliferation, respectively. Micro indentation was conducted to quantify the mechanical integrity of the native and acellular dura graft. The findings indicate that the acellular porcine dura graft creates a beneficial setting for infiltrating rat fibroblast cells. Cell viability, proliferation, and micro indentation results on the acellular grafts are comparable with the native control porcine dura tissue. In conclusion, the porcine scaffold material showed increased cell viability at each time point evaluated. The sustained mechanical response and favorable viability of the cells on the decellularized grafts provide promising insight into the potential use of porcine dura in clinical cranial dura mater graft applications.

Engineering the multiscale complexity of vascular networks.

The survival of vertebrate organisms depends on highly regulated delivery of oxygen and nutrients through vascular networks that pervade nearly all tissues in the body. Dysregulation of these vascular networks is implicated in many common human diseases such as hypertension, coronary artery disease, diabetes and cancer. Therefore, engineers have sought to create vascular networks within engineered tissues for applications such as regenerative therapies, human disease modelling and pharmacological testing. Yet engineering vascular networks has historically remained difficult, owing to both incomplete understanding of vascular structure and technical limitations for vascular fabrication. This Review highlights the materials advances that have enabled transformative progress in vascular engineering by ushering in new tools for both visualizing and building vasculature. New methods such as bioprinting, organoids and microfluidic systems are discussed, which have enabled the fabrication of 3D vascular topologies at a cellular scale with lumen perfusion. These approaches to vascular engineering are categorized into technology-driven and nature-driven approaches. Finally, the remaining knowledge gaps, emerging frontiers and opportunities for this field are highlighted, including the steps required to replicate the multiscale complexity of vascular networks found in nature.