Increased macrophage phagocytic activity with TLR9 agonist conjugation of an anti- Borrelia burgdorferi monoclonal antibody.

Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing.

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection. Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadin's involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes ΔinlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP ΔLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Cell-stretching devices: advances and challenges in biomedical research and live-cell imaging.

Basic human functions such as breathing and digestion require mechanical stretching of cells and tissues. However, when it comes to laboratory experiments, the mechanical stretching that cells experience in the body is not often replicated, limiting the biomimetic nature of the studies and the relevance of results. Herein, we establish the importance of mechanical stretching during in vitro investigations by reviewing seminal works performed using cell-stretching platforms, highlighting important outcomes of these works as well as the engineering characteristics of the platforms used. Emphasis is placed on the compatibility of cell-stretching devices (CSDs) with live-cell imaging as well as their limitations and on the research advancements that could arise from live-cell imaging performed during cell stretching.

Integration of Extracellular Matrices into Organ-on-Chip Systems.

The extracellular matrix (ECM) is a complex, dynamic network present within all tissues and organs that not only acts as a mechanical support and anchorage point but can also direct fundamental cell behavior, function, and characteristics. Although the importance of the ECM is well established, the integration of well-controlled ECMs into Organ-on-Chip (OoC) platforms remains challenging and the methods to modulate and assess ECM properties on OoCs remain underdeveloped. In this review, current state-of-the-art design and assessment of in vitro ECM environments is discussed with a focus on their integration into OoCs. Among other things, synthetic and natural hydrogels, as well as polydimethylsiloxane (PDMS) used as substrates, coatings, or cell culture membranes are reviewed in terms of their ability to mimic the native ECM and their accessibility for characterization. The intricate interplay among materials, OoC architecture, and ECM characterization is critically discussed as it significantly complicates the design of ECM-related studies, comparability between works, and reproducibility that can be achieved across research laboratories. Improving the biomimetic nature of OoCs by integrating properly considered ECMs would contribute to their further adoption as replacements for animal models, and precisely tailored ECM properties would promote the use of OoCs in mechanobiology.

Alterations of baroreflex sensitivity after carotid endarterectomy according to the preoperative carotid plaque echogenicity.

OBJECTIVE: Baroreflex sensitivity is lower in patients with echogenic carotid plaques compared with patients with echolucent ones. The purpose of our study was to compare the baroreflex function after carotid endarterectomy (CEA) between patients with different plaque echogenicity. METHOD: Spontaneous baroreflex sensitivity (sBRS), heart rate, and systolic and diastolic arterial pressure were calculated in 51 patients with a severe carotid stenosis (70%-99%) 24 hours before CEA, as well as 24 and 48 hours after CEA. Carotid plaque echogenicity was graded from 1 to 4 according to Gray-Weale classification, after duplex examination, and the patients were divided into two groups: the echolucent (grade 1 or 2) and the echogenic (grade 3 or 4). RESULTS: The postoperative mean systolic arterial pressure values in all 51 patients at 24 and 48 hours (143.2 and 135.5 mm Hg, respectively) were found to be significantly increased compared with the preoperative value (132.5 mm Hg; x2=32, P<.001). Mean sBRS value, in all patients, was significantly reduced postoperatively to 2.1 ms mm Hg(-1), from the mean preoperative value, 3.7 ms mm Hg(-1), independently of plaque echogenicity. Twenty patients (39%) were included in the echolucent group and 31 (61%) in the echogenic. The two groups had significant differences in two parameters: the rate of diabetes mellitus and the rate of symptomatic plaques. After adjusting the two groups for these differences, we found that the preoperative difference in sBRS between the two groups (F[1,51]=11, P<.003) was eliminated 24 and 48 hours after CEA (F[1,51]=.007, P<.9 and F[1,51]=.4, P<.5 for 24 and 48 hours, respectively). CONCLUSIONS: Before the removal of carotid atheroma, baroreflex sensitivity, which is a well established cardiovascular risk factor, seems to be affected by carotid plaque echogenicity. However, CEA has as a result a similar baroreflex response in all patients, regardless of plaque echogenicity, implying no association of plaque morphology and postoperative baroreflex sensitivity.

Mechanical Forces Govern Interactions of Host Cells with Intracellular Bacterial Pathogens.

To combat infectious diseases, it is important to understand how host cells interact with bacterial pathogens. Signals conveyed from pathogen to host, and vice versa, may be either chemical or mechanical. While the molecular and biochemical basis of host-pathogen interactions has been extensively explored, relatively less is known about mechanical signals and responses in the context of those interactions. Nevertheless, a wide variety of bacterial pathogens appear to have developed mechanisms to alter the cellular biomechanics of their hosts in order to promote their survival and dissemination, and in turn many host responses to infection rely on mechanical alterations in host cells and tissues to limit the spread of infection. In this review, we present recent findings on how mechanical forces generated by host cells can promote or obstruct the dissemination of intracellular bacterial pathogens. In addition, we discuss how in vivo extracellular mechanical signals influence interactions between host cells and intracellular bacterial pathogens. Examples of such signals include shear stresses caused by fluid flow over the surface of cells and variable stiffness of the extracellular matrix on which cells are anchored. We highlight bioengineering-inspired tools and techniques that can be used to measure host cell mechanics during infection. These allow for the interrogation of how mechanical signals can modulate infection alongside biochemical signals. We hope that this review will inspire the microbiology community to embrace those tools in future studies so that host cell biomechanics can be more readily explored in the context of infection studies.

Spatiotemporal characterization of endothelial cell motility and physical forces during exposure to Borrelia burgdorferi.

Cell motility and biomechanics are critical in various (patho)physiological processes, including the regulation of vascular barrier integrity, which can be subverted by bacterial pathogens. Here, we present a protocol on how to expose endothelial cells (ECs) to vector-borne Borrelia burgdorferi (Bb) and characterize EC kinematics and dynamics during exposure to live or heat-inactivated Bb through traction force and monolayer stress microscopy. Modifications to this protocol may be necessary for studying how different cell types interact with Bb or other microorganisms. For complete details on the use and execution of this protocol, please refer to Yuste et al. (2022).1.

A Stiff Extracellular Matrix Favors the Mechanical Cell Competition that Leads to Extrusion of Bacterially-Infected Epithelial Cells.

Cell competition refers to the mechanism whereby less fit cells ("losers") are sensed and eliminated by more fit neighboring cells ("winners") and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected "loser" cells by uninfected "winner" cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations.

Borrelia burgdorferi modulates the physical forces and immunity signaling in endothelial cells.

Borrelia burgdorferi (Bb), a vector-borne bacterial pathogen and the causative agent of Lyme disease, can spread to distant tissues in the human host by traveling in and through monolayers of endothelial cells (ECs) lining the vasculature. To examine whether Bb alters the physical forces of ECs to promote its dissemination, we exposed ECs to Bb and observed a sharp and transient increase in EC traction and intercellular forces, followed by a prolonged decrease in EC motility and physical forces. All variables returned to baseline at 24 h after exposure. RNA sequencing analysis revealed an upregulation of innate immune signaling pathways during early but not late Bb exposure. Exposure of ECs to heat-inactivated Bb recapitulated only the early weakening of EC mechanotransduction. The differential responses to live versus heat-inactivated Bb indicate a tight interplay between innate immune signaling and physical forces in host ECs and suggest their active modulation by Bb.

The role of carotid plaque echogenicity in baroreflex sensitivity.

OBJECTIVE: The baroreflex sensitivity is impaired in patients with carotid atherosclerosis. The purpose of our study was to assess the impact of carotid plaque echogenicity on the baroreflex function in patients with significant carotid atherosclerosis, who have not undergone carotid surgery. METHOD: Spontaneous baroreflex sensitivity (sBRS) was estimated in 45 patients with at least a severe carotid stenosis (70%-99%). sBRS calculation was performed noninvasively, with the spontaneous sequence method, based on indirectly estimated central blood pressures from radial recordings. This method failed in three patients due to poor-quality recordings, and eventually 42 patients were evaluated. After carotid duplex examination, carotid plaque echogenicity was graded from 1 to 4 according to Gray-Weale classification and the patients were divided into two groups: the echolucent group (grades 1 and 2) and the echogenic group (grades 3 and 4). RESULTS: Sixteen patients (38%) and 26 patients (62%) were included in the echolucent and echogenic group, respectively. Diabetes mellitus was observed more frequently among echolucent plaques (χ(2) = 8.0; P < .004), while those plaques were also more commonly symptomatic compared with echogenic atheromas (χ(2) = 8.5; P < .003). Systolic arterial pressure, diastolic arterial pressure, and heart rate were similar in the two groups. Nevertheless, the mean value of baroreflex sensitivity was found to be significantly lower in the echogenic group (2.96 ms/mm Hg) compared with the echolucent one (5.0 ms/mm Hg), (F [1, 42] = 10.1; P < .003). CONCLUSIONS: These findings suggest that echogenic plaques are associated with reduced baroreflex function compared with echolucent ones. Further investigation is warranted to define whether such an sBRS impairment could be responsible for cardiovascular morbidity associated with echogenic plaques.

Volume measurement and biophysical characterization of mounds in epithelial monolayers after intracellular bacterial infection.

Mechanical forces are important in (patho)physiological processes, including how host epithelial cells interact with intracellular bacterial pathogens. As these pathogens disseminate within host epithelial monolayers, large mounds of infected cells are formed due to the forceful action of surrounding uninfected cells, limiting bacterial spread across the basal cell monolayer. Here, we present a protocol for mound volume measurement and biophysical characterization of mound formation. Modifications to this protocol may be necessary for studying different host cell types or pathogenic organisms. For complete details on the use and execution of this protocol, please refer to Bastounis et al. (2021).

Mechanical competition triggered by innate immune signaling drives the collective extrusion of bacterially infected epithelial cells.

Intracellular pathogens alter their host cells' mechanics to promote dissemination through tissues. Conversely, host cells may respond to the presence of pathogens by altering their mechanics to limit infection. Here, we monitored epithelial cell monolayers infected with intracellular bacterial pathogens, Listeria monocytogenes or Rickettsia parkeri, over days. Under conditions in which these pathogens trigger innate immune signaling through NF-κB and use actin-based motility to spread non-lytically intercellularly, we found that infected cell domains formed three-dimensional mounds. These mounds resulted from uninfected cells moving toward the infection site, collectively squeezing the softer and less contractile infected cells upward and ejecting them from the monolayer. Bacteria in mounds were less able to spread laterally in the monolayer, limiting the growth of the infection focus, while extruded infected cells underwent cell death. Thus, the coordinated forceful action of uninfected cells actively eliminates large domains of infected cells, consistent with this collective cell response representing an innate immunity-driven process.

Subendothelial stiffness alters endothelial cell traction force generation while exerting a minimal effect on the transcriptome.

Endothelial cells respond to changes in subendothelial stiffness by altering their migration and mechanics, but whether those responses are due to transcriptional reprogramming remains largely unknown. We measured traction force generation and also performed gene expression profiling for two endothelial cell types grown in monolayers on soft or stiff matrices: primary human umbilical vein endothelial cells (HUVEC) and immortalized human microvascular endothelial cells (HMEC-1). Both cell types respond to changes in subendothelial stiffness by increasing the traction stresses they exert on stiffer as compared to softer matrices, and exhibit a range of altered protein phosphorylation or protein conformational changes previously implicated in mechanotransduction. However, the transcriptome has only a minimal role in this conserved biomechanical response. Only few genes were differentially expressed in each cell type in a stiffness-dependent manner, and none were shared between them. In contrast, thousands of genes were differentially regulated in HUVEC as compared to HMEC-1. HUVEC (but not HMEC-1) upregulate expression of TGF-ß2 on stiffer matrices, and also respond to application of exogenous TGF-ß2 by enhancing their endogenous TGF-ß2 expression and their cell-matrix traction stresses. Altogether, these findings provide insights into the relationship between subendothelial stiffness, endothelial mechanics and variation of the endothelial cell transcriptome, and reveal that subendothelial stiffness, while critically altering endothelial cells' mechanical behavior, minimally affects their transcriptome.

Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin.

Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been studied previously, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens is hitherto unexplored. To address this open question, we manufactured hydrogels of varying physiologically relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm) and found that adhesion of Lm to host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types' responses to matrix stiffness have been characterized, how adherent cells' susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.