Combinatorial Single-Cell Analyses of Granulocyte-Monocyte Progenitor Heterogeneity Reveals an Early Uni-potent Neutrophil Progenitor.

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs.

Visualising cancer in 3D: 3-Dimensional Tissue Imaging for management of cutaneous basal cell carcinoma.

Surgical management of basal cell carcinoma (BCC) typically involves surgical excision with post-operative margin assessment using the bread-loafing technique; or gold-standard Mohs micrographic surgery (MMS), where margins are iteratively examined for residual cancer after tumour removal, with additional excisions performed upon detecting residual tumour at margins. There is limited sampling of resection margins with bread loafing, with detection of positive margins 44% of the time using 2 mm intervals. To resolve this, we have developed three-dimensional (3D) Tissue Imaging for: (1) complete examination of cancer margins and (2) detection of tumour proximity to nerves and blood vessels. 3D Tissue optical clearing with a light sheet imaging protocol was developed for margin assessment in two datasets assessed by two independent evaluators: (1) 48 samples from 29 patients with varied BCC subtypes, sizes and pigmentation levels; (2) 32 samples with matching Mohs' surgeon reading of tumour margins using two-dimensional haematoxylin & eosin-stained sections. The 3D Tissue Imaging protocol permits a complete examination of deeper and peripheral margins. Two independent evaluators achieved negative predictive values of 92.3% and 88.24% with 3D Tissue Imaging. Images obtained from 3D Tissue Imaging recapitulates histological features of BCC, such as nuclear crowding, palisading and retraction clefting and provides a 3D context for recognising normal skin adnexal structures. Concurrent immunofluorescence labelling of nerves and blood vessels allows visualisation of structures closer to tumour-positive regions, which may have a higher risk for neural and vascular infiltration. Together, this method provides more information in a 3D spatial context, enabling better cancer management by clinicians.

Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice.

Tissue-resident macrophages in white adipose tissue (WAT) dynamically adapt to the metabolic changes of their microenvironment that are often induced by excess energy intake. Currently, the exact contribution of these macrophages in obesity-driven WAT remodeling remains controversial. Here, using a transgenic CD169-DTR mouse strain, we provide new insights into the interplay between CD169+ adipose tissue macrophages (ATMs) and their surrounding WAT microenvironment. Using targeted in vivo ATM ablation followed by transcriptional and metabolic WAT profiling, we found that ATMs protect WAT from the excessive pathological remodeling that occurs during obesity. As obesity progresses, ATMs control not only vascular integrity, adipocyte function, and lipid and metabolic derangements but also extracellular matrix accumulation and resultant fibrosis in the WAT. The protective role of ATMs during obesity-driven WAT dysfunction supports the notion that ATMs represent friends, rather than foes, as has previously assumed.

Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer.

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either C3aR or C5aR1 by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.

Tyrosine Dephosphorylation of ASC Modulates the Activation of the NLRP3 and AIM2 Inflammasomes.

The inflammasome is an intracellular multi-protein complex that orchestrates the release of the pro-inflammatory cytokines IL-1ß and IL-18, and a form of cell death known as pyroptosis. Tyrosine phosphorylation of the inflammasome sensors NLRP3, AIM2, NLRC4, and the adaptor protein, apoptosis-associated speck-like protein (ASC) has previously been demonstrated to be essential in the regulation of the inflammasome. By using the pharmacological protein tyrosine phosphatase (PTPase) inhibitor, phenylarsine oxide (PAO), we have demonstrated that tyrosine dephosphorylation is an essential step for the activation of the NLRP3 and AIM2 inflammasomes in human and murine macrophages. We have also shown that PTPase activity is required for ASC nucleation leading to caspase-1 activation, IL-1ß, and IL-18 processing and release, and cell death. Furthermore, by site-directed mutagenesis of ASC tyrosine residues, we have identified the phosphorylation of tyrosine Y60 and Y137 of ASC as critical for inflammasome assembly and function. Therefore, we report that ASC tyrosine dephosphorylation and phosphorylation are crucial events for inflammasome activation.

The Inflammasome Adaptor ASC Intrinsically Limits CD4+ T-Cell Proliferation to Help Maintain Intestinal Homeostasis.

The inflammasome is a multi-protein complex that mediates proteolytic cleavage and release of the pro-inflammatory cytokines IL-1ß and IL-18, and pyroptosis-a form of cell death induced by various pathogenic bacteria. Apoptosis-associated speck-like protein containing a CARD (ASC) has a pivotal role in inflammasome assembly and activation. While ASC function has been primarily implicated in innate immune cells, its contribution to lymphocyte biology is unclear. Here we report that ASC is constitutively expressed in naïve CD4+ T cells together with the inflammasome sensor NLRP3 and caspase-1. When adoptively transferred in immunocompromised Rag1-/- mice, Asc-/- CD4+ T cells exacerbate T-cell-mediated autoimmune colitis. Asc-/- CD4+ T cells exhibit a higher proliferative capacity in vitro than wild-type CD4+ T cells. The increased expansion of Asc-/- CD4+ T cells in vivo correlated with robust TCR-mediated activation, inflammatory activity, and higher metabolic profile toward a highly glycolytic phenotype. These findings identify ASC as a crucial intrinsic regulator of CD4+ T-cell expansion that serves to maintain intestinal homeostasis.

Salmonella typhimurium-induced IL-1 release from primary human monocytes requires NLRP3 and can occur in the absence of pyroptosis.

Large molecular complexes known as inflammasomes regulate the release of IL-1ß from immune cells in response to infection and injury. Salmonella typhimurium infection is reported to activate NLRP3 and NLRC4 inflammasomes which are subsequently involved in pyroptosis of the cell and pathogen clearance. However, the response to S. typhimurium in primary human monocytes has not been studied in detail. The aim of this study was to investigate the effect of S. typhimurium on inflammasomes in primary human monocytes. Much of the previous research in the field has been conducted in murine models and human THP-1 cells, which may not reflect the responses of primary human monocytes. Here, we report that inhibiting NLRP3 with the selective inhibitor MCC950, blocked release of IL-1ß and the related cytokine IL-1α from primary human monocytes in response to S. typhimurium. Additionally, under these conditions S. typhimurium-induced IL-1 release occurred independently of pyroptosis. We propose that IL-1ß release without pyroptosis may occur in early-recruited monocytes to regulate a maximal innate immune response to Salmonella infection, allowing a sustained inflammatory signal. This insight into the mechanisms involved in IL-1 release from primary human monocytes highlights major differences between immune cell types, and the defences they employ during bacterial infection.

NLRP10 Enhances CD4+ T-Cell-Mediated IFNγ Response via Regulation of Dendritic Cell-Derived IL-12 Release.

NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated a Nlrp10-/- mouse to delineate the role of NLRP10 in the host immune response and found that Nlrp10-/- dendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4+ T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation, Nlrp10-/- DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evident in vivo and affected IFNγ production by CD4+ T cells. Upon Mycobacterium tuberculosis (Mtb) infection, Nlrp10-/- mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against Mtb.

Reduction of Photoluminescence Quenching by Deuteration of Ytterbium-Doped Amorphous Carbon-Based Photonic Materials.

In situ Yb-doped amorphous carbon thin films were grown on Si substrates at low temperatures (<200 °C) by a simple one-step RF-PEMOCVD system as a potential photonic material for direct integration with Si CMOS back end-of-line processing. Room temperature photoluminescence around 1 µm was observed via direct incorporation of optically active Yb3+ ions from the selected Yb(fod)3 metal-organic compound. The partially fluorinated Yb(fod)3 compound assists the suppression of photoluminescence quenching by substitution of C-H with C-F bonds. A four-fold enhancement of Yb photoluminescence was demonstrated via deuteration of the a-C host. The substrate temperature greatly influences the relative deposition rate of the plasma dissociated metal-organic species, and hence the concentration of the various elements. Yb and F incorporation are promoted at lower substrate temperatures, and suppressed at higher substrate temperatures. O concentration is slightly elevated at higher substrate temperatures. Photoluminescence was limited by the concentration of Yb within the film, the concentration of Yb ions in the +3 state, and the relative amount of quenching due to the various de-excitation pathways associated with the vibrational modes of the host a-C network. The observed wide full-width-at-half-maximum photoluminescence signal is a result of the variety of local bonding environments due to the a-C matrix, and the bonding of the Yb3+ ions to O and/or F ions as observed in the X-ray photoelectron spectroscopy analyses.

Erbium-Doped Amorphous Carbon-Based Thin Films: A Photonic Material Prepared by Low-Temperature RF-PEMOCVD.

The integration of photonic materials into CMOS processing involves the use of new materials. A simple one-step metal-organic radio frequency plasma enhanced chemical vapor deposition system (RF-PEMOCVD) was deployed to grow erbium-doped amorphous carbon thin films (a-C:(Er)) on Si substrates at low temperatures (<200 °C). A partially fluorinated metal-organic compound, tris(6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5- octanedionate) Erbium(+III) or abbreviated Er(fod)3, was incorporated in situ into a-C based host. Six-fold enhancement of Er room-temperature photoluminescence at 1.54 µm was demonstrated by deuteration of the a-C host. Furthermore, the effect of RF power and substrate temperature on the photoluminescence of a-C:D(Er) films was investigated and analyzed in terms of the film structure. Photoluminescence signal increases with increasing RF power, which is the result of an increase in [O]/[Er] ratio and the respective erbium-oxygen coordination number. Moreover, photoluminescence intensity decreases with increasing substrate temperature, which is attributed to an increased desorption rate or a lower sticking coefficient of the fluorinated fragments during film growth and hence [Er] decreases. In addition, it is observed that Er concentration quenching begins at ~2.2 at% and continues to increase until 5.5 at% in the studied a-C:D(Er) matrix. This technique provides the capability of doping Er in a vertically uniform profile.