Faculty Bibliography

Development, morphogenesis, immune system function, and cancer metastasis rely on the ability of cells to move through diverse tissues. To dissect migratory cell behavior in vivo, we developed cell type-specific imaging and perturbation techniques for Drosophila primordial germ cells (PGCs). We find that PGCs use global, retrograde cortical actin flows for orientation and propulsion during guided developmental homing. PGCs use RhoGEF2, a RhoA-specific RGS-RhoGEF, as a dose-dependent regulator of cortical flow through a feedback loop requiring its conserved PDZ and PH domains for membrane anchoring and local RhoA activation. This feedback loop is regulated for directional migration by RhoGEF2 availability and requires AMPK rather than canonical Gα_12/13 signaling. AMPK multisite phosphorylation of RhoGEF2 near a conserved EB1 microtubule-binding SxIP motif releases RhoGEF2 from microtubule-dependent inhibition. Thus, we establish the mechanism by which global cortical flow and polarized RhoA activation can be dynamically adapted during natural cell navigation in a changing environment.

Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions-lacking somatic gonadal cell coverage of germ cells-are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of gene expression, yet their contribution to immune regulation in humans remains poorly understood. Here, we report that the primate-specific lncRNA CHROMR is induced by influenza A virus and SARS-CoV-2 infection and coordinates the expression of interferon-stimulated genes (ISGs) that execute antiviral responses. CHROMR depletion in human macrophages reduces histone acetylation at regulatory regions of ISG loci and attenuates ISG expression in response to microbial stimuli. Mechanistically, we show that CHROMR sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional corepressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Consequently, CHROMR expression is essential to restrict viral infection of macrophages. Our findings identify CHROMR as a key arbitrator of antiviral innate immune signaling in humans.

OBJECTIVE:After injury, humans and other mammals heal by forming fibrotic scar tissue with diminished function, and this healing process involves the dynamic interplay between resident cells within the skin and cells recruited from the circulation. Recent studies have provided mounting evidence that external mechanical forces stimulate intracellular signaling pathways to drive fibrotic processes. INNOVATION/METHODS:While most studies have focused on studying mechanotransduction in fibroblasts, recent data suggest that mechanical stimulation may also shape the behavior of immune cells, referred to as "mechano-immunomodulation". However, the effect of mechanical strain on myeloid cell recruitment and differentiation remains poorly understood and has never been investigated at the single cell level. APPROACH/METHODS:In this study, we utilized a three-dimensional (3D) in vitro culture system that permits the precise manipulation of mechanical strain applied to cells. We cultured myeloid cells and used single cell RNA-sequencing to interrogate the effects of strain on myeloid differentiation and transcriptional programming. RESULTS:Our data indicate that myeloid cells are indeed mechanoresponsive, with mechanical stress influencing myeloid differentiation. Mechanical strain also upregulated a cascade of inflammatory chemokines, most notably from the Ccl family. CONCLUSION/CONCLUSIONS:Further understanding of how mechanical stress affects myeloid cells in conjunction with other cell types in the complicated, multicellular milieu of wound healing may lead to novel insights and therapies for the treatment of fibrosis.

Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants^1-3. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space^4-9. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline-proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.

Transforming growth factor-beta (TGFβ) is released from cells as part of a trimeric latent complex consisting of TGFβ, the TGFβ propeptides, and either a latent TGFβ binding protein (LTBP) or glycoprotein-A repetitions predominant (GARP) protein. LTBP1 and 3 modulate latent TGFβ function with respect to secretion, matrix localization, and activation and, therefore, are vital for the proper function of the cytokine in a number of tissues. TGFβ modulates stem cell differentiation into adipocytes (adipogenesis), but the potential role of LTBPs in this process has not been studied. We observed that 72 h post adipogenesis initiation Ltbp1, 2, and 4 expression levels decrease by 74-84%, whereas Ltbp3 expression levels remain constant during adipogenesis. We found that LTBP3 silencing in C3H/10T1/2 cells reduced adipogenesis, as measured by the percentage of cells with lipid vesicles and the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ). Lentiviral mediated expression of an Ltbp3 mRNA resistant to siRNA targeting rescued the phenotype, validating siRNA specificity. Knockdown (KD) of Ltbp3 expression in 3T3-L1, M2, and primary bone marrow stromal cells (BMSC) indicated a similar requirement for Ltbp3. Epididymal and inguinal white adipose tissue fat pad weights of Ltbp3^-/- mice were reduced by 62% and 57%, respectively, compared to wild-type mice. Inhibition of adipogenic differentiation upon LTBP3 loss is mediated by TGFβ, as TGFβ neutralizing antibody and TGFβ receptor I kinase blockade rescue the LTBP3 KD phenotype. These results indicate that LTBP3 has a TGFβ-dependent function in adipogenesis both in vitro and possibly in vivo.

Response to cancer immunotherapy in primary versus metastatic disease has not been well-studied. We found primary pancreatic ductal adenocarcinoma (PDA) is responsive to diverse immunotherapies whereas liver metastases are resistant. We discovered divergent immune landscapes in each compartment. Compared to primary tumor, liver metastases in both mice and humans are infiltrated by highly anergic T cells and MHCII^loIL10⁺ macrophages that are unable to present tumor-antigen. Moreover, a distinctive population of CD24⁺CD44^-CD40^- B cells dominate liver metastases. These B cells are recruited to the metastatic milieu by Muc1^hiIL18^hi tumor cells, which are enriched >10-fold in liver metastases. Recruited B cells drive macrophage-mediated adaptive immune-tolerance via CD200 and BTLA. Depleting B cells or targeting CD200/BTLA enhanced macrophage and T-cell immunogenicity and enabled immunotherapeutic efficacy of liver metastases. Our data detail the mechanistic underpinnings for compartment-specific immunotherapy-responsiveness and suggest that primary PDA models are poor surrogates for evaluating immunity in advanced disease.

OBJECTIVE:mice. METHODS:mice were fed a typical Western-style diet for 22 weeks and injected with a nontargeting locked nucleic acid (LNA; LNA control) or miR-148a LNA (LNA 148a) for the last 10 weeks. At the end of the treatment, the mice were sacrificed, and circulating lipids, hepatic gene expression, and atherosclerotic lesions were analyzed. RESULTS:Examination of atherosclerotic lesions revealed a significant reduction in plaque size, with marked remodeling of the lesions toward a more stable phenotype. Mechanistically, miR-148a levels influenced macrophage cholesterol efflux and the inflammatory response. Suppression of miR-148a in murine primary macrophages decreased mRNA levels of proinflammatory M1-like markers (Nos2, Il6, Cox2, and Tnf) and increased the expression of anti-inflammatory genes (Arg1, Retlna, and Mrc1). CONCLUSIONS:Therapeutic silencing of miR148a mitigated the progression of atherosclerosis and promoted plaque stability. The antiatherogenic effect of miR-148a antisense therapy is likely mediated by the anti-inflammatory effects observed in macrophages treated with miR-148 LNA and independent of significant changes in circulating low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

PURPOSE/OBJECTIVE:Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to an imbalance in bone metabolism leading to osteoporosis. More recently, the use of SSRIs in murine models has been shown to delay bone healing both in vivo and in vitro by decreasing the osteoblastic differentiation and mineralization. The purpose of this study was to evaluate whether or not chronic use of SSRI's in nonunion patients increases their time to union after surgical intervention. METHODS:We retrospectively analyzed 343 patients in a nonunion database to determine which patients were on SSRI medication. Of these patients, 139 could be contacted and of those 102 were not taking SSRIs and 37 were taking SSRIs. Patient's time to union from nonunion surgical intervention between each cohort at our institution was recorded as the primary outcome. Patient's medical comorbidities that could affect union rates such as diabetes and smoking status were also noted. Baseline Short Musculoskeletal Function Assessment (SMFA) index for bother and function were recorded from the time of nonunion surgery as well as last follow-up. RESULTS:Compared to recent census data, we found significantly more patients in the nonunion cohort using SSRIs (26.6%) than patients in the general population using any type of antidepressant (11%). There was no significant difference in the patients' baseline characteristics other than patients on SSRI treatment had a higher body mass index (BMI) and age (p = 0.048 and p = 0.043, respectively). There was no significant difference noted in the fracture types (p = 0.2063). Patients on SSRIs had a higher SMFA bother index and function index on follow-up (p = 0.0103, p = 0.0147). Patients in the SSRI group had a mean time to union from nonunion surgery of 6.1 months compared to 6.0 in patients without SSRI usage (p = 0.74). These did not reach statistical significance when subcohort analysis for long bone fractures was performed for the femur, tibia, and humerus. CONCLUSION/CONCLUSIONS:To our knowledge, this is the first clinical study to investigate the effects of SSRIs on fracture healing. While in vivo and in vitro murine models have shown that SSRIs can have a deleterious effect on osteoblastic activity, our retrospective analysis did not show a significant difference in time to union between patients with chronic SSRI use and patients who have not been on SSRIs. However, this investigation did show a higher incidence of SSRI use in the nonunion cohort when compared to the general population. In the context of the recent animal model study, this may point to a negative effect of SSRI use on the acute fracture healing process.

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.