Complement Receptor 3 Mediates HIV-1 Transcytosis across an Intact Cervical Epithelial Cell Barrier: New Insight into HIV Transmission in Women

In women, the lower female reproductive tract is the primary site for HIV infection. How HIV traverses the epithelium to infect CD4 T cells in the submucosa is ill-defined.

Neuronal apoptosis drives remodeling states of microglia and shifts in survival pathway dependence

Microglia serve critical remodeling roles that shape the developing nervous system, responding to the changing neural environment with phagocytosis or soluble factor secretion. Recent single-cell sequencing (scRNAseq) studies have revealed the context-dependent diversity in microglial properties and gene expression, but the cues promoting this diversity are not well defined. Here, we ask how interactions with apoptotic neurons shape microglial state, including lysosomal and lipid metabolism gene expression and independence from Colony-stimulating factor 1 receptor (CSF1R) for survival. Using early postnatal mouse retina, a CNS region undergoing significant developmental remodeling, we performed scRNAseq on microglia from mice that are wild-type, lack neuronal apoptosis (Bax KO), or are treated with CSF1R inhibitor (PLX3397). We find that interactions with apoptotic neurons drives multiple microglial remodeling states, subsets of which are resistant to CSF1R inhibition. We find that TAM receptor Mer and complement receptor 3 are required for clearance of apoptotic neurons, but that Mer does not drive expression of remodeling genes. We show TAM receptor Axl is negligible for phagocytosis or remodeling gene expression but is consequential for microglial survival in the absence of CSF1R signaling. Thus, interactions with apoptotic neurons shift microglia towards distinct remodeling states and through Axl, alters microglial dependence on survival pathway, CSF1R.

Inside-out Signalling From Aminopeptidase N (CD13) To Complement Receptor 3 (CR3, CD11b/CD18)

Upon ligand engagement, certain receptors can activate an integrin through a mechanism called inside-out signalling. This phenomenon prepares the cell for the next steps of the process it will perform. CR3 (Complement receptor 3), the most abundant β2 integrin in monocytes and macrophages, and CD13 (aminopeptidase N) are two immune receptors with overlapping activities: adhesion, phagocytosis of opsonized particles, and respiratory burst induction. They can be found together in functional signalling microdomains, or lipid rafts, on the surface of human leukocytes. Thus, given their common functions, shared physical location and the fact that some phagocytic and adhesion receptors activate a selection of integrins, we hypothesized that CD13 could activate CR3 through an inside-out signalling mechanism. To test this hypothesis, we first ascertained the activation of CR3 after CD13 crosslinking in human monocyte-derived macrophages. We used an integrated analysis of bioinformatics and experimental data to suggest two possible signalling cascades that could explain the phenomenon. Finally, we show that the non-receptor tyrosine kinase Syk is a key attenuator of this pathway. Our results demonstrated that, even in the absence of canonical signalling motifs, and despite having a noticeably short cytoplasmic tail (7-10 amino acids), CD13 was capable of triggering an inside-out signalling cascade, adding a new function to those already known for this moonlighting protein.

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble β-Glucan

β‐Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8–P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

Endothelial Progenitor Cell Transplantation Attenuated Synaptic Loss by Enhancing CR3 Dependent Microglial Phagocytosis in Mice

Background: Endothelial progenitor cell (EPC) transplantation has been shown to have therapeutic effects in cerebral ischemia. However, whether the therapeutic effect of EPCs is a result of the modulation of microglia activity remain elusive. Methods: Adult male mice (n=184) underwent 90 minute-middle cerebral artery occlusion and EPCs were transplanted into the peri-infarct region immediately after the surgery. Microglia migration and phagocytosis were evaluated in the ischemic brain in vivo and underwent oxygen-glucose-deprivation culture condition in vitro. Complement receptor 3 was examined in ischemic brain and cultured primary microglia. Complement receptor 3 agonist leukadherin-1 was intraperitoneally injected to mice immediately after ischemia to imitate the EPC effect. Expression of synapse remodeling related synaptophysin and PSD-95 proteins was detected in the EPC and leukadherin-1 treated mice, separately. Results: EPC transplantation increased the number of microglia in the peri-infarct region of the brain at 3 days after focal ischemia (p<0.05). The ability of phagocytizing apoptotic cells of microglia was higher in EPCs transplanted group at 3 days after ischemia compared to the controls (p<0.05). In vitro study showed that cultured microglia displayed a higher migration (p<0.05) and phagocytosis ability (p<0.05) under the stimulation of EPC conditioned medium or cultured EPCs compared to the controls. Complement receptor 3 expression in the ischemic mouse brain with EPC transplantation (p<0.05), and primary microglia treated by EPC conditioned medium or cultured EPCs was up-regulated (p<0.05). Leukadherin-1 reduced brain atrophy volume at 14 days (p<0.05) and ameliorated neurological deficiency during 14 days after cerebral ischemia (p<0.05). Both EPC transplantation and leukadherin-1 injection increased synaptophysin (p<0.05) and PSD-95 expressions (p<0.05) at 14 days after focal ischemia. Conclusion: We concluded that EPC transplantation promoted regulating complement receptor 3 mediated microglial phagocytosis at acute phase, and subsequently benefited for attenuating synaptic loss at the recovery phase of ischemic stroke, which provided a novel therapeutic mechanism of EPC for cerebral ischemia.

Mac-1 Receptor Clustering Initiates Production of Pro-Inflammatory, Antibacterial Extracellular Vesicles From Neutrophils

Depending on the prevailing environmental conditions, neutrophilic granulocytes release extracellular vesicles (EV) which have either anti-inflammatory effects on other neutrophils or pro-inflammatory and antibacterial effects. In the present study we investigated the molecular mechanisms underlying the biogenesis of functionally heterogenic EVs. We show that selective stimulation of Mac-1 integrin (complement receptor 3) by specific ligands initiates the generation of EVs which are able to impair bacterial growth and to induce the secretion of the pro-inflammatory cytokine IL-8 (aEV). However, direct Mac-1 stimulation results in aEV release only if neutrophils were activated on ligand coated surfaces whereas soluble ligands are ineffective. Using total internal reflection fluorescence (TIRF) microcopy, an increased clustering of Mac-1 molecules could be visualized in neutrophils added to C3bi coated surfaces; moreover antibody induced cluster formation triggers aEV release as well. Mac-1 induced production of aEV apparently necessitates a strong calcium signal as it fully depends on the presence of extracellular calcium. However, initiation of a strong calcium signal by an ionophore only results the generation of EV devoid of any antibacterial or pro-inflammatory effect. Our results thus demonstrate that stimulation and clustering of Mac-1 is necessary and sufficient for initiation of aEV biogenesis. In contrast, an intracellular calcium signal is necessary but by itself not sufficient for the production of antibacterial and pro-inflammatory EVs.