Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

In matters of the heart, (cellular) communication is key.

To maintain cardiac output, the failing heart undergoes significant remodeling. However, the mechanisms regulating this remain unclear. In this issue of Immunity, Zaman et al. and Wong, Mohan, and Kopecky et al. uncover an interaction between resident cardiac macrophages and cardiomyocytes governing this process.

Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming.

Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity.

Osteopontin characterizes bile duct-associated macrophages and correlates with liver fibrosis severity in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which pharmacological treatment options are currently unavailable. PSC is strongly associated with colitis and a disruption of the gut-liver axis, and macrophages are involved in the pathogenesis of PSC. However, how gut-liver interactions and specific macrophage populations contribute to PSC is incompletely understood. APPROACH AND RESULTS: We investigated the impact of cholestasis and colitis on the hepatic and colonic microenvironment, and performed an in-depth characterization of hepatic macrophage dynamics and function in models of concomitant cholangitis and colitis. Cholestasis-induced fibrosis was characterized by depletion of resident KCs, and enrichment of monocytes and monocyte-derived macrophages (MoMFs) in the liver. These MoMFs highly express triggering-receptor-expressed-on-myeloid-cells-2 ( Trem2 ) and osteopontin ( Spp1 ), markers assigned to hepatic bile duct-associated macrophages, and were enriched around the portal triad, which was confirmed in human PSC. Colitis induced monocyte/macrophage infiltration in the gut and liver, and enhanced cholestasis-induced MoMF- Trem2 and Spp1 upregulation, yet did not exacerbate liver fibrosis. Bone marrow chimeras showed that knockout of Spp1 in infiltrated MoMFs exacerbates inflammation in vivo and in vitro , while monoclonal antibody-mediated neutralization of SPP1 conferred protection in experimental PSC. In human PSC patients, serum osteopontin levels are elevated compared to control, and significantly increased in advanced stage PSC and might serve as a prognostic biomarker for liver transplant-free survival. CONCLUSIONS: Our data shed light on gut-liver axis perturbations and macrophage dynamics and function in PSC and highlight SPP1/OPN as a prognostic marker and future therapeutic target in PSC.

Kupffer Cells Contested as Early Drivers in the Pathogenesis of Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) is an idiopathic chronic immune-mediated cholestatic liver disease characterized by fibro-inflammatory bile duct strictures, progressive hepatobiliary fibrosis, and gut-liver axis disruption. The pathophysiology of PSC remains insufficiently characterized, which hampers the development of effective therapies. Hepatic macrophages (MFs) such as Kupffer cells (KCs) are implicated in PSC pathogenesis, but their exact role is unclear. Using the latest markers to discriminate resident KCs (ResKCs) from their monocyte-derived counterparts (MoKCs), and two models of intrahepatic and extrahepatic cholestasis, respectively, this study showed that CLEC4F+TIM4+ ResKCs were depleted after chronic cholestatic liver injury. The infiltrating CLEC4F+TIM4- MoKCs were already enriched during the acute phase of PSC. Transcriptional profiling of hepatic MF subsets during early cholestatic injury indicated that ResKCs were indeed activated and that MoKCs expressed higher levels of pro-inflammatory and proliferative markers compared with those of ResKCs. As indicated in experiments with Clec4fDTR transgenic mice, conditional depletion of KCs, before and during early cholestasis induction, had no effect on the composition of the hepatic myeloid cell pool following injury progression and did not affect disease outcomes. Taken together, these results provide new insights into the heterogeneity of the MF pool during experimental PSC and evidence that depletion of resident and activated KCs during sclerosing cholangitis does not affect disease outcome in mice.

Inhibitor activity of Lactiplantibacillus plantarum LP5 on thermotolerant campylobacter with different biofilm-forming capacities.

AIMS: To evaluate the biofilm-forming capacity of thermotolerant Campylobacter (TC) strains from poultry production and to analyse the inhibitory capacity of Lactiplantibacillus plantarum LP5 against TC on different materials. METHODS AND RESULTS: Biofilm-forming capacity by Campylobacter jejuni and Campylobacter coli was analysed by cell adhesion in polystyrene plates. TC were classified as non-biofilm-forming (NBF, 1.3%), weak biofilm-forming (WBF, 68.4%), moderate biofilm-forming (MBF, 27.6%), and strong biofilm-forming (SBF, 2.7%). The inhibitory capacity of L. plantarum LP5 against TC was tested on stainless-steel, nylon, aluminium, and glass disks (treated group) and compared with biofilm-forming TC (control group). Lactiplantibacillus plantarum LP5 was inoculated, and then TC. Biofilm was removed in both experimental groups and TC and LP5 bacterial counts were performed. The L. plantarum LP5 presence reduced the formation of TC biofilm (P < 0.001). The material type and strain category influenced biofilm formation, with stainless-steel and the SBF strain being the material and TC having the highest adhesion (P < 0.001). Lactiplantibacillus plantarum LP5 formed a similar biofilm on all materials (P = 0.823). CONCLUSIONS: This trial showed very promising results; L. plantarum LP5 could be incorporated as a bio-protector of TC on different surfaces.

PATTERN OF A PRIMARY B-CELL LYMPHOMA IN ULNAR NERVE: INTRANEURAL OR EXTRANEURAL.

Primary lymphomas of peripheral nerves (PLPNs) are extremely rare and most commonly reported in lumbar nerves and have been found in only five cases in the upper extremities. We describe two patterns of presentation focusing on clinical, radiological, and pathological findings of two patients affected by primary multifocal lymphoma of the ulnar nerve without systemic involvement or other medical conditions. We report a case of extraneural lymphoma in a 72-years-old (patient #1) and a case of intraneural lymphoma in a 45-years old woman (Patient #2). Magnetic resonance imaging and ultrasound findings were similar to Peripheral Nerve Sheath Tumors (PNST). Surgical exploration and excision were performed. Morpho pathological results revealed in both cases a diffuse large B-cell non-Hodgkin lymphoma. In patient #1, the disease relapsed after only 4 months with brachial plexus involvement. The patient died about 10 months after the onset of symptoms. Patient #2 did not have post-surgical sensory or motor deficit and follow up at 6 years did not show recurrence or any other localizations. PLPN is a rare and challenging condition and is frequently misdiagnosed. PLPNs could have an intraneural or an extraneural pattern. As peripheral neuropathy may be caused by a nervous involvement by a lymphoma, in patients with atypical lesions, a complete preoperative imaging should be acquired.

Modulating hepatic macrophages with annexin A1 in non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) and associated end-stage liver disease is a growing cause of concern throughout the Western world. It constitutes a significant clinical burden for which therapeutic approaches are very limited. Over the last years, considerable attention has therefore been paid to identifying potential therapeutic strategies to reduce this burden. Annexin A1 (AnxA1), a calcium-phospholipid binding protein, has been proposed to be a negative regulator of inflammation in the context of NASH. In a recent publication, Gadipudi, Ramavath, Provera et al. investigated the therapeutic potential of Annexin A1 treatment in preventing the progression of NASH. They demonstrate that treatment of mice with NASH with recombinant human AnxA1 can reduce inflammation and fibrosis without affecting steatosis or metabolic syndrome. This was proposed to be achieved through the modulation of the macrophage populations present in the liver. Here, we discuss the main findings of this work and raise some outstanding questions regarding the possible mechanisms involved and the functions of distinct macrophage populations in NASH.

From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics.

Serological tests detect antibodies generated by infection or vaccination, and are indispensable tools along different phases of a pandemic, from early monitoring of pathogen spread up to seroepidemiological studies supporting immunization policies. This work discusses the development of an accurate and affordable COVID-19 antibody test, from production of a recombinant protein antigen up to test validation and economic analysis. We first developed a cost-effective, scalable technology to produce SARS-COV-2 spike protein and then used this antigen to develop an enzyme-linked immunosorbent assay (ELISA). A receiver operator characteristic (ROC) analysis allowed optimizing the cut-off and confirmed the high accuracy of the test: 98.6% specificity and 95% sensitivity for 11+ days after symptoms onset. We further showed that dried blood spots collected by finger pricking on simple test strips could replace conventional plasma/serum samples. A cost estimate was performed and revealed a final retail price in the range of one US dollar, reflecting the low cost of the ELISA test platform and the elimination of the need for venous blood sampling and refrigerated sample handling in clinical laboratories. The presented workflow can be completed in 4 months from first antigen expression to final test validation. It can be applied to other pathogens and in future pandemics, facilitating reliable and affordable seroepidemiological surveillance also in remote areas and in low-income countries.

Familial primary ovarian insufficiency associated with an SYCE1 point mutation: defective meiosis elucidated in humanized mice.

More than 50% of cases of primary ovarian insufficiency (POI) and nonobstructive azoospermia in humans are classified as idiopathic infertility. Meiotic defects may relate to at least some of these cases. Mutations in genes coding for synaptonemal complex (SC) components have been identified in humans, and hypothesized to be causative for the observed infertile phenotype. Mutation SYCE1 c.721C>T (former c.613C>T)-a familial mutation reported in two sisters with primary amenorrhea-was the first such mutation found in an SC central element component-coding gene. Most fundamental mammalian oogenesis events occur during the embryonic phase, and eventual defects are identified many years later, thus leaving few possibilities to study the condition's etiology and pathogenesis. Aiming to validate an approach to circumvent this difficulty, we have used the CRISPR/Cas9 technology to generate a mouse model with an SYCE1 c.721C>T equivalent genome alteration. We hereby present the characterization of the homozygous mutant mice phenotype, compared to their wild type and heterozygous littermates. Our results strongly support a causative role of this mutation for the POI phenotype in human patients, and the mechanisms involved would relate to defects in homologous chromosome synapsis. No SYCE1 protein was detected in homozygous mutants and Syce1 transcript level was highly diminished, suggesting transcript degradation as the basis of the infertility mechanism. This is the first report on the generation of a humanized mouse model line for the study of an infertility-related human mutation in an SC component-coding gene, thus representing a proof of principle.

Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis.

The discovery of a large number of long noncoding RNAs (lncRNAs), and the finding that they may play key roles in different biological processes, have started to provide a new perspective in the understanding of gene regulation. It has been shown that the testes express the highest amount of lncRNAs among different vertebrate tissues. However, although some studies have addressed the characterization of lncRNAs along spermatogenesis, an exhaustive analysis of the differential expression of lncRNAs at its different stages is still lacking. Here, we present the results for lncRNA transcriptome profiling along mouse spermatogenesis, employing highly pure flow sorted spermatogenic stage-specific cell populations, strand-specific RNAseq, and a combination of up-to-date bioinformatic pipelines for analysis. We found that the vast majority of testicular lncRNA genes are expressed at post-meiotic stages (i.e. spermiogenesis), which are characterized by extensive post-transcriptional regulation. LncRNAs at different spermatogenic stages shared common traits in terms of transcript length, exon number, and biotypes. Most lncRNAs were lincRNAs, followed by a high representation of antisense (AS) lncRNAs. Co-expression analyses showed a high correlation along the different spermatogenic stage transitions between the expression patterns of AS lncRNAs and their overlapping protein-coding genes, raising possible clues about lncRNA-related regulatory mechanisms. Interestingly, we observed the co-localization of an AS lncRNA and its host sense mRNA in the chromatoid body, a round spermatids-specific organelle that has been proposed as a reservoir of RNA-related regulatory machinery. An additional, intriguing observation is the almost complete lack of detectable expression for Y-linked testicular lncRNAs, despite that a high number of lncRNA genes are annotated for this chromosome.

Axonal GABAA receptors depolarize presynaptic terminals and facilitate transmitter release in cerebellar Purkinje cells.

KEY POINTS: GABAA receptors have been described in the axonal compartment of neurons; contrary to dendritic GABAA receptors, axonal GABAA receptors usually induce depolarizing responses. In this study we describe the presence of functional axonal GABAA receptors in cerebellar Purkinje cells by using a combination of direct patch-clamp recordings from the axon terminals and laser GABA photolysis. In Purkinje cells, axonal GABAA receptors are depolarizing and induce an increase in neurotransmitter release that results in a change of short-term synaptic plasticity. These results contribute to our understanding of the cellular mechanisms of action of axonal GABAA receptors and highlight the importance of the presynaptic compartment in neuronal computation. ABSTRACT: In neurons of the adult brain, somatodendritic GABAA receptors (GABAA Rs) mediate fast synaptic inhibition and play a crucial role in synaptic integration. GABAA Rs are not only present in the somatodendritic compartment, but also in the axonal compartment where they modulate action potential (AP) propagation and transmitter release. Although presynaptic GABAA Rs have been reported in various brain regions, their mechanisms of action and physiological roles remain obscure, particularly at GABAergic boutons. Here, using a combination of direct whole-bouton or perforated patch-clamp recordings and local GABA photolysis in single axonal varicosities of cerebellar Purkinje cells, we investigate the subcellular localization and functional role of axonal GABAA Rs both in primary cultures and acute slices. Our results indicate that presynaptic terminals of PCs carry GABAA Rs that behave as auto-receptors; their activation leads to a depolarization of the terminal membrane after an AP due to the relatively high cytoplasmic Cl- concentration in the axon, but they do not modulate the AP itself. Paired recordings from different terminals of the same axon show that the GABAA R-mediated local depolarizations propagate substantially to neighbouring varicosities. Finally, the depolarization mediated by presynaptic GABAA R activation augmented Ca2+ influx and transmitter release, resulting in a marked effect on short-term plasticity. Altogether, our results reveal a mechanism by which presynaptic GABAA Rs influence neuronal computation.

Transcriptome analysis of highly purified mouse spermatogenic cell populations: gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage.

BACKGROUND: Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis. RESULTS: We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation. CONCLUSIONS: This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.

Vascular responsiveness determined by near-infrared spectroscopy measures of oxygen saturation.

Vascular impairments at the macro- and microcirculatory levels are associated with increased risk for cardiovascular disease. Flow-mediated dilation (FMD) is currently the most widely used method for non-invasive assessment of vascular endothelial function. Recently, near-infrared spectroscopy (NIRS)-derived measures of tissue oxygen saturation (StO2) have been used to characterize the dynamic response of local tissue perfusion to a brief period of ischaemia. The purpose of the present study was to establish correlations between the reperfusion rate of StO2 and FMD. Ultrasound-derived FMD was quantified after 5 min of distal cuff occlusion of the popliteal artery in 20 healthy young men (26 ± 3 years old). Triplicate measurements of end-diastolic arterial diameter were made every 15 s after cuff release, and FMD response was calculated as the greatest percentage change in diameter from baseline (%FMD). The StO2 was measured using NIRS throughout the duration of each test. Two consecutive FMD tests were performed, separated by 30 min of rest, and were averaged for %FMD and StO2. The %FMD was significantly correlated with the reperfusion slope of StO2 after cuff release (slope 2 StO2; r = 0.63, P = 0.003). In conclusion, the present study established a correlation between slope 2 StO2 and %FMD in healthy young men. These data suggest that NIRS-derived slope 2 StO2 can be used as a measure of vascular endothelial function.

MTCH2 is differentially expressed in rat testis and mainly related to apoptosis of spermatocytes.

MTCH2 has been described in liver as a protein involved in the intrinsic apoptotic pathway, although new evidence also associates this protein with cellular metabolism. In this work, the expression of MTCH2 in testis (an organ in which high levels of apoptosis normally take place as part of the spermatogenic process) is analyzed in rat, both at the mRNA and at the protein levels. Our results showed that MTCH2 was highly expressed in testis compared with other tissues and was differentially expressed according to developmental stage and testicular cell type. Protein expression was initially detected during the first spermatogenic wave at the time of meiosis onset and its levels increased in adulthood, with the highest expression levels being detected in meiotic prophase I. Specific differences in MTCH2 expression levels at the various stages of the adult seminiferous epithelium were also observed. Co-staining with TUNEL revealed a differential MTCH2 staining pattern in TUNEL-positive cells, mainly in dying primary spermatocytes, i.e., meiotic prophase I cells. Furthermore, upon mild hyperthermia (treatment shown to increase apoptosis in testis), MTCH2 levels rose concomitantly with a massive appearance of TUNEL-positive cells within the seminiferous tubules; these cells exhibited a differential MTCH2 distribution. Thus, MTCH2 is related to testicular apoptosis, especially during meiotic prophase.

Readily releasable pool of synaptic vesicles measured at single synaptic contacts.

To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

Synthesis and biological evaluation of bis-CNB-GABA, a photoactivatable neurotransmitter with low receptor interference and chemical two-photon uncaging properties.

Photoactivatable "caged" neurotransmitters allow optical control of neural tissue with high spatial and temporal precision. However, the development of caged versions of the chief vertebrate inhibitory neurotransmitter, γ-amino butyric acid (GABA), has been limited by the propensity of caged GABAs to interact with GABA receptors. We describe herein the synthesis and application of a practically useful doubly caged GABA analog, termed bis-α-carboxy-2-nitrobenzyl-GABA (bis-CNB-GABA). Uncaging of bis-CNB-GABA evokes inward GABAergic currents in cerebellar molecular layer interneurons with rise times of 2 ms, comparable to flash duration. Response amplitudes depend on the square of flash intensity, as expected for a chemical two-photon uncaging effect. Importantly, prior to uncaging, bis-CNB-GABA is inactive at the GABAA receptor, evoking no changes in holding current in voltage-clamped neurons and showing an IC50 of at least 2.5 mM as measured using spontaneous GABAergic synaptic currents. Bis-CNB-GABA is stable in solution, with an estimated half-life of 98 days in the light. We expect that bis-CNB-GABA will prove to be an effective tool for high-resolution chemical control of brain circuits.

Learning by observation in children with autism spectrum disorder.

BACKGROUND: Observing another person performing a complex action accelerates the observer's acquisition of the same action and limits the time-consuming process of learning by trial and error. Learning by observation requires specific skills such as attending, imitating and understanding contingencies. Individuals with autism spectrum disorder (ASD) exhibit deficits in these skills. METHOD: The performance of 20 ASD children was compared with that of a group of typically developing (TD) children matched for chronological age (CA), IQ and gender on tasks of learning of a visuomotor sequence by observation or by trial and error. Acquiring the correct sequence involved three phases: a detection phase (DP), in which participants discovered the correct sequence and learned how to perform the task; an exercise phase (EP), in which they reproduced the sequence until performance was error free; and an automatization phase (AP), in which by repeating the error-free sequence they became accurate and speedy. RESULTS: In the DP, ASD children were impaired in detecting a sequence by trial and error only when the task was proposed as first, whereas they were as efficient as TD children in detecting a sequence by observation. In the EP, ASD children were as efficient as TD children. In the AP, ASD children were impaired in automatizing the sequence. Although the positive effect of learning by observation was evident, ASD children made a high number of imitative errors, indicating marked tendencies to hyperimitate. CONCLUSIONS: These findings demonstrate the imitative abilities of ASD children although the presence of imitative errors indicates an impairment in the control of imitative behaviours.