A role for Sfrp2 in cardiomyogenesis in vivo

Cardiomyogenesis, the process by which the body generates cardiomyocytes, is poorly understood. We have recently shown that Sfrp2 promotes cardiomyogenesis in vitro. The objective of this study was to determine if Sfrp2 would similarly promote cardiomyogenesis in vivo. To test this hypothesis, we tracked multipotent cKit(+) cells in response to Sfrp2 treatment. In control adult mice, multipotent cKit(+) cells typically differentiated into endothelial cells but not cardiomyocytes. In contrast, Sfrp2 switched the fate of these cells. Following Sfrp2 injection, multipotent cKit(+) cells differentiated solely into cardiomyocytes. Sfrp2-derived cardiomyocytes integrated into the myocardium and exhibited identical physiological properties to preexisting native cardiomyocytes. The ability of Sfrp2 to promote cardiomyogenesis was further supported by tracking EdU-labeled cells. In addition, Sfrp2 did not promote the formation of new cardiomyocytes when the cKit(+) cell population was selectively ablated in vivo using a diphtheria toxin receptor–diphtheria toxin model. Notably, Sfrp2-induced cardiomyogenesis was associated with significant functional improvements in a cardiac injury model. In summary, our study further demonstrates the importance of Sfrp2 in cardiomyogenesis.

Microglial ablation does not affect opioid-induced hyperalgesia in rodents

Opioids are the frontline analgesics in pain management. However, chronic use of opioid analgesics causes paradoxical pain that contributes to the decrease of their efficacy in pain control and the escalation of dose in long-term management of pain. The underling pathogenic mechanism is not well understood. Microglia have been commonly thought to play a critical role in the expression of opioid-induced hyperalgesia (OIH) in animal models. We performed microglial ablation experiments using either a genetic (CD11b-diphtheria toxin receptor transgenic mouse) or pharmacological (colony-stimulating factor 1 receptor inhibitor PLX5622) approaches. Surprisingly, ablating microglia using these specific and effective approaches did not cause detectable impairment in the expression of hyperalgesia induced by morphine. We confirmed this conclusion with behavioral test of mechanical and thermal hyperalgesia, in male and female mice, and with different species (mouse and rat). These findings raise caution about the widely assumed contribution of microglia to the development of OIH.

CD206+ macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model

In endometriosis, M2 MΦs are dominant in endometriotic lesions, but the actual role of M2 MΦ is unclear. CD206 positive (+) MΦ is classified in one of M2 type MΦs and are known to produce cytokines and chemokines. In the present study, we used CD206 diphtheria toxin receptor mice, which enable to deplete CD206+ cells with diphtheria toxin (DT) in an endometriosis mouse model. The depletion of CD206+ MΦ decreased the total weight of endometriotic-like lesions significantly (p < 0.05). In the endometriotic-like lesions in the DT group, a lower proliferation of endometriotic cells and the decrease of angiogenesis were observed. In the lesions, the mRNA levels of VEGFA and TGFβ1, angiogenic factors, in the DT group significantly decreased to approximately 50% and 30% of control, respectively. Immunohistochemical study revealed the expressions of VEGFA and an endothelial cell marker CD31 in lesions of the DT group, were dim compared to those in control. Also, the number of TGFβ1 expressing MΦ was significantly reduced compared to control. These data suggest that CD206+ MΦ promotes the formation of endometriotic-like lesions by inducing angiogenesis around the lesions.

Underestimated Peripheral Effects Following Pharmacological and Conditional Genetic Microglial Depletion

Microglia, predominant parenchymal resident macrophages in the central nervous system (CNS), are crucial players in neurodevelopment and CNS homeostasis. In disease conditions, pro-inflammatory microglia predominate over their regulatory counterparts, and are thus a potential immunotherapeutic target. It has been well documented that microglia can be effectively depleted using both conditional genetic Cx3cr1Cre-diphtheria toxin receptor (DTR)/diphtheria toxin subunit A (DTA) animal models and pharmacological colony-stimulating factor 1 receptor (CSF1R) inhibitors. Recent advances using these approaches have expanded our knowledge of the multitude of tasks conducted by microglia in both homeostasis and diseases. Importantly, experimental microglial depletion has been proven to exert neuroprotective effects in an increasing number of disease models, mostly explained by reduced neuroinflammation. However, the comprehensive effects of additional targets such as circulating monocytes and peripheral tissue macrophages during microglial depletion periods have not been investigated widely, and for those studies addressing the issue the conclusions are mixed. In this study, we demonstrate that experimental microglial depletion using both Cx3cr1CreER/+Rosa26DTA/+ mice and different doses of CSF1R inhibitor PLX3397 exert crucial influences on circulating monocytes and peripheral tissue macrophages. Our results suggest that effects on peripheral immunity should be considered both in interpretation of microglial depletion studies, and especially in the potential translation of microglial depletion and replacement therapies.

An IgA mimicry of IgG that binds polymeric immunoglobulin receptor for mucosa transcytosis

Most pathogens establish infection through mucosa, where secretory immunoglobulin A (sIgA) plays an ‘immune exclusion’ role in humoral defense. Extravasation of intravenously (i.v.) administrated therapeutic immunoglobulin G (IgG) mainly relies on convection and/or neonatal Fc receptor-mediated transcytosis from circulation into interstitial space. Active transport of interstitial IgG further across epithelium into mucosa, like sIgA, is a much desired feature for the next generation of therapeutic antibodies, especially for anti-infection purposes. For the first time, we report the engineering of an IgA mimicry of IgG, with its Fc portion in fusion with the 18-aa tail piece (tp) of sIgA and the J chain, possessing sIgA’s full binding activity towards polymeric immunoglobulin receptor that mediates mucosa transcytosis. In a diphtheria toxin receptor (DTR) knockin mouse model, i.v. injected anti-diphtheria toxin (DT) IgG(tp)J protected DTR+ cells from deletion upon DT injection. The compact design of IgG(tp)J opens new revenues for more effective therapeutic IgG mimicking some of the important biological functions of IgA.

Ly6CLo Monocyte/Macrophages are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis

Venous thrombosis (VT) resolution is a complex process, resembling sterile wound healing. Infiltrating blood-derived monocyte/macrophages (Mo/MΦs) are essential for the regulation of inflammation in tissue repair. These cells differentiate into inflammatory (CD11b+Ly6CHi) or proreparative (CD11b+Ly6CLo) subtypes. Previous studies have shown that infiltrating Mo/MΦs are important for VT resolution, but the precise roles of different Mo/MΦs subsets are not well understood. Utilizing murine models of stasis and stenosis inferior vena cava thrombosis in concert with a Mo/MΦ depletion model (CD11b-diphtheria toxin receptor [DTR]-expressing mice), we examined the effect of Mo/MΦ depletion on thrombogenesis and VT resolution. In the setting of an 80 to 90% reduction in circulating CD11b+Mo/MΦs, we demonstrated that Mo/MΦs are not essential for thrombogenesis, with no difference in thrombus size, neutrophil recruitment, or neutrophil extracellular traps found. Conversely, CD11b+Mo/MΦ are essential for VT resolution. Diphtheria toxoid (DTx)-mediated depletion after thrombus creation depleted primarily CD11b+Ly6CLo Mo/MΦs and resulted in larger thrombi. DTx-mediated depletion did not alter CD11b+Ly6CHi Mo/MΦ recruitment, suggesting a protective effect of CD11b+Ly6CLo Mo/MΦs in VT resolution. Confirmatory Mo/MΦ depletion with clodronate lysosomes showed a similar phenotype, with failure to resolve VT. Adoptive transfer of CD11b+Ly6CLo Mo/MΦs into Mo/MΦ-depleted mice reversed the phenotype, restoring normal thrombus resolution. These findings suggest that CD11b+Ly6CLo Mo/MΦs are essential for normal VT resolution, consistent with the known proreparative function of this subset, and that further study of Mo/MΦ subsets may identify targets for immunomodulation to accelerate and improve thrombosis resolution.

Distinct Role of CD11b+Ly6G−Ly6C− Myeloid-Derived Cells on the Progression of the Primary Tumor and Therapy-Associated Recurrent Brain Tumor

Myeloid-derived cells have been implicated as playing essential roles in cancer therapy, particularly in cancer immunotherapy. Most studies have focused on either CD11b+Ly6G+Ly6C+ granulocytic or polymorphonuclear myeloid-derived suppressor cells (G-MDSCs or PMN-MDSCs) or CD11b+Ly6G−Ly6C+ monocytic MDSCs (M-MDSCs), for which clear roles have been established. On the other hand, CD11b+Ly6G−Ly6C− myeloid-derived cells (MDCs) have been less well studied. Here, the CD11b-diphtheria toxin receptor (CD11b-DTR) transgenic mouse model was used to evaluate the role of CD11b+ myeloid-derived cells in chemotherapy for an orthotopic murine astrocytoma, ALTS1C1. Using this transgenic mouse model, two injections of diphtheria toxin (DT) could effectively deplete CD11b+Ly6G−Ly6C− MDCs while leaving CD11b+Ly6G+Ly6C+ PMN-MDSCs and CD11b+Ly6G−Ly6C+ M-MDSCs intact. Depletion of CD11b+Ly6G−Ly6C− MDCs in mice bearing ALTS1C1-tk tumors and receiving ganciclovir (GCV) prolonged the mean survival time for mice from 30.7 to 37.8 days, but not the controls, while the effectiveness of temozolomide was enhanced. Mechanistically, depletion of CD11b+Ly6G−Ly6C− MDCs blunted therapy-induced increases in tumor-associated macrophages (TAMs) and compromised therapy-elicited angiogenesis. Collectively, our findings suggest that CD11b+Ly6G−Ly6C− MDCs could be manipulated to enhance the efficacy of chemotherapy for brain tumors. However, our study also cautions that the timing of any MDC manipulation may be critical to achieve the best therapeutic result.

Enteroendocrine and tuft cells support Lgr5 stem cells on Paneth cell depletion

Cycling intestinal Lgr5+stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

Islet macrophages are associated with islet vascular remodeling and compensatory hyperinsulinemia during diabetes

β-Cells respond to peripheral insulin resistance by first increasing circulating insulin during diabetes. Islet remodeling supports this compensation, but its drivers remain poorly understood. Infiltrating macrophages have been implicated in late-stage type 2 diabetes, but relatively little is known on islet resident macrophages, especially during compensatory hyperinsulinemia. We hypothesized that islet resident macrophages would contribute to islet vascular remodeling and hyperinsulinemia during diabetes, the failure of which results in a rapid progression to frank diabetes. We used chemical (clodronate), genetics (CD169-diphtheria toxin receptor mice), or antibody-mediated (colony-stimulating factor 1 receptor α) macrophage ablation methods in diabetic (db/db) and diet-induced models of compensatory hyperinsulinemia to investigate the role of macrophages in islet remodeling. We transplanted islets devoid of macrophages into naïve diabetic mice and assessed the impact on islet vascularization. With the use of the above methods, we showed that macrophage depletion significantly and consistently compromised islet remodeling in terms of size, vascular density, and insulin secretion capacity. Depletion of islet macrophages reduced VEGF-A secretion in both human and mouse islets ex vivo, and this functionally translated to delayed revascularization upon transplantation in vivo. We revealed that islet resident macrophages were associated with islet remodeling and increased insulin secretion during diabetes. This suggests utility in harnessing islet macrophages during this phase to promote islet vascularization, remodeling, and insulin secretion.

Natural Killer cells dampen the pathogenic features of recall responses to influenza infection

Despite evidence of augmented Natural Killer (NK) cell responses after influenza vaccination, the role of these cells in vaccine-induced immunity remains unclear. Here, we hypothesized that NK cells might increase viral clearance but possibly at the expense of increased severity of pathology. On the contrary, we found that NK cells serve a homeostatic role during influenza virus infection of vaccinated mice, allowing viral clearance with minimal pathology. Using a diphtheria toxin receptor transgenic mouse model, we were able to specifically deplete NKp46+ NK cells through the administration of diphtheria toxin. Using this model, we assessed the effect of NK cell depletion prior to influenza challenge in vaccinated and unvaccinated mice. NK-depleted, vaccinated animals lost significantly more weight after viral challenge than vaccinated NK intact animals, indicating that NK cells ameliorate disease in vaccinated animals. However, there was also a significant reduction in viral load in NK-depleted, unvaccinated animals indicating that NK cells also constrain viral clearance. Depletion of NK cells after vaccination, but 21 days before infection, did not affect viral clearance or weight loss - indicating that it is the presence of NK cells during the infection itself that promotes homeostasis. Further work is needed to identify the mechanism(s) by which NK cells regulate adaptive immunity in influenza-vaccinated animals to allow efficient and effective virus control whilst simultaneously minimizing inflammation and pathology.