Absence of Aquaporin-4 (AQP4) Prolongs the Presence of a CD11c+ Microglial Population during Postnatal Corpus Callosum Development.

Aquaporin-4 (AQP4) expression is associated with the development of congenital hydrocephalus due to its structural role in the ependymal membrane. Gene expression analysis of periaqueductal tissue in AQP4-knockout (KO) mice at 11 days of age (P11) showed a modification in ependymal cell adhesion and ciliary protein expression that could alter cerebrospinal fluid homeostasis. A microglial subpopulation of CD11c+ cells was overexpressed in the periaqueductal tissue of mice that did not develop hydrocephalus, suggesting a possible protective effect. Here, we verified the location of this CD11c+ expression in the corpus callosum (CC) and cerebellum of AQP4-KO mice and analysed its time course. Immunofluorescence labelling of the CD11c protein in the CC and cerebellum of WT and KO animals at P3, P5, P7 and P11 confirmed an expanded presence of these cells in both tissues of the KO animal; CD11c+ cells appeared at P3 and reached a peak at P11, whereas in the WT animal, they appeared at P5, reached their peak at P7 and were undetectable by P11. The gene expression analysis in the CC samples at P11 confirmed the presence of CD11c+ microglial cells in this tissue. Among the more than 4000 overexpressed genes, Spp1 stood out with the highest differential gene expression (≅600), with other genes, such as Gpnmb, Itgax, Cd68 and Atp6v0d2, also identified as overexpressed. Therefore, CD11c+ cells appear to be necessary for normal corpus callosum development during postnatal life, and the absence of AQP4 prolonged its expression in this tissue.

PIKfyve, expressed by CD11c-positive cells, controls tumor immunity.

Cancer treatment continues to shift from utilizing traditional therapies to targeted ones, such as protein kinase inhibitors and immunotherapy. Mobilizing dendritic cells (DC) and other myeloid cells with antigen presenting and cancer cell killing capacities is an attractive but not fully exploited approach. Here, we show that PIKFYVE is a shared gene target of clinically relevant protein kinase inhibitors and high expression of this gene in DCs is associated with poor patient response to immune checkpoint blockade (ICB) therapy. Genetic and pharmacological studies demonstrate that PIKfyve ablation enhances the function of CD11c+ cells (predominantly dendritic cells) via selectively altering the non-canonical NF-κB pathway. Both loss of Pikfyve in CD11c+ cells and treatment with apilimod, a potent and specific PIKfyve inhibitor, restrained tumor growth, enhanced DC-dependent T cell immunity, and potentiated ICB efficacy in tumor-bearing mouse models. Furthermore, the combination of a vaccine adjuvant and apilimod reduced tumor progression in vivo. Thus, PIKfyve negatively regulates the function of CD11c+ cells, and PIKfyve inhibition has promise for cancer immunotherapy and vaccine treatment strategies.

Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.

Regulation of B-cell function and expression of CD11c, T-bet, and FcRL5 in response to different activation signals.

CD11c, FcRL5, or T-bet are commonly expressed by B cells expanding during inflammation, where they can make up >30% of mature B cells. However, the association between the proteins and differentiation and function in the host response remains largely unclear. We have assessed the co-expression of CD11c, T-bet, and FcRL5 in an in vitro B-cell culture system to determine how stimulation via the BCR, toll-like receptor 9 (TLR9), and different cytokines influence CD11c, T-bet, and FcRL5 expression. We observed different expression dynamics for all markers, but a largely overlapping regulation of CD11c and FcRL5 in response to BCR and TLR9 activation, while T-bet was strongly dependent on IFN-γ signaling. Investigating plasma cell differentiation and APC functions, there was no association between marker expression and antibody secretion or T-cell help. Rather the functions were associated with TLR9-signalling and B-cell-derived IL-6 production, respectively. These results suggest that the expression of CD11c, FcRL5, and T-bet and plasma cell differentiation and improved APC functions occur in parallel and are regulated by similar activation signals, but they are not interdependent.

Lactate Dehydrogenase-Elevating Virus Infection Inhibits MOG Peptide Presentation by CD11b+CD11c+ Dendritic Cells in a Mouse Model of Multiple Sclerosis.

Infections may affect the course of autoimmune inflammatory diseases of the central nervous system (CNS), such as multiple sclerosis (MS). Infections with lactate dehydrogenase-elevating virus (LDV) protected mice from developing experimental autoimmune encephalomyelitis (EAE), a mouse counterpart of MS. Uninfected C57BL/6 mice immunized with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) experienced paralysis and lost weight at a greater rate than mice who had previously been infected with LDV. LDV infection decreased the presentation of the MOG peptide by CD11b+CD11c+ dendritic cells (DC) to pathogenic T lymphocytes. When comparing non-infected mice to infected mice, the histopathological examination of the CNS showed more areas of demyelination and CD45+ and CD3+, but not Iba1+ cell infiltration. These results suggest that the protective effect of LDV infection against EAE development is mediated by a suppression of myelin antigen presentation by a specific DC subset to autoreactive T lymphocytes. Such a mechanism might contribute to the general suppressive effect of infections on autoimmune diseases known as the hygiene hypothesis.

Spatiotemporal dynamics of the CD11c+ microglial population in the mouse brain and spinal cord from developmental to adult stages.

CD11c-positive (CD11c+) microglia have attracted considerable attention because of their potential implications in central nervous system (CNS) development, homeostasis, and disease. However, the spatiotemporal dynamics of the proportion of CD11c+ microglia in individual CNS regions are poorly understood. Here, we investigated the proportion of CD11c+ microglia in six CNS regions (forebrain, olfactory bulb, diencephalon/midbrain, cerebellum, pons/medulla, and spinal cord) from the developmental to adult stages by flow cytometry and immunohistochemical analyses using a CD11c reporter transgenic mouse line, Itgax-Venus. We found that the proportion of CD11c+ microglia in total microglia varied between CNS regions during postnatal development. Specifically, the proportion was high in the olfactory bulb and cerebellum at postnatal day P(4) and P7, respectively, and approximately half of the total microglia were CD11c+. The proportion declined sharply in all regions to P14, and the low percentage persisted over P56. In the spinal cord, the proportion of CD11c+ microglia was also high at P4 and declined to P14, but increased again at P21 and thereafter. Interestingly, the distribution pattern of CD11c+ microglia in the spinal cord markedly changed from gray matter at P4 to white matter at P21. Collectively, our findings reveal the differences in the spatiotemporal dynamics of the proportion of CD11c+ microglia among CNS regions from early development to adult stages in normal mice. These findings improve our understanding of the nature of microglial heterogeneity and its dynamics in the CNS.

Correlation of Professional Antigen-Presenting Tbet+CD11c+ B Cells With Bone Destruction in Untreated Rheumatoid Arthritis.

OBJECTIVE: Subsets of CD21-/low memory B cells (MBCs), including double-negative (DN, CD27-IgD-) and Tbet+CD11c+ cells, are expanded in chronic inflammatory diseases. In rheumatoid arthritis (RA), CD21-/low MBCs correlate with joint destruction. However, whether this is due to the Tbet+CD11c+ subset, its function and pathogenic contribution to RA are unknown. This study aims to investigate the association between CD21-/lowTbet+CD11c+ MBCs and joint destruction as well as other clinical parameters and to elucidate their functional properties in patients with untreated RA (uRA). METHODS: Clinical observations were combined with flow cytometry (n = 36) and single-cell RNA sequencing (scRNA-seq) and V(D)J sequencing (n = 4) of peripheral blood (PB) MBCs from patients with uRA. The transcriptome of circulating Tbet+CD11c+ MBCs was compared with scRNA-seq data of synovial B cells. In vitro coculture of Tbet+CD11c+ B cells with T cells was used to assess costimulatory capacity. RESULTS: CD21-/lowTbet+CD11c+ MBCs in PB correlated with bone destruction but no other clinical parameters analyzed. The Tbet+CD11c+ MBCs have undergone clonal expansion and express somatically mutated V genes. Gene expression analysis of these cells identified a unique signature of more than 150 up-regulated genes associated with antigen presentation functions, including B cell receptor activation and clathrin-mediated antigen internalization; regulation of actin filaments, endosomes, and lysosomes; antigen processing, loading, presentation, and costimulation; a transcriptome mirrored in their synovial tissue counterparts. In vitro, Tbet+CD11c+ B cells induced retinoic acid receptor-related orphan nuclear receptor γT expression in CD4+ T cells, thereby polarizing to Th17 cells, a T cell subset critical for osteoclastogenesis and associated with bone destruction. CONCLUSION: This study suggests that Tbet+CD11c+ MBCs contribute to the pathogenesis of RA by promoting bone destruction through antigen presentation, T cell activation, and Th17 polarization.

Indirect CD4+ T cell protection against mouse gamma-herpesvirus infection via interferon gamma.

CD4+ T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4+ T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4+ T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4+ T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c+ cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII+ CD11c+ antigen-presenting cells is key to the CD4+ T cell/NK cell protection axis. By contrast, CD8+ T cell control of AEC1 infection appeared to operate independently. IMPORTANCE: CD4+ T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4+ T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c+ dendritic cells. This mechanism of CD4+ T cell control releases the need for the direct engagement of CD4+ T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens.

Nuclear Factor κB Signaling Deficiency in CD11c-Expressing Phagocytes Mediates Early Inflammatory Responses and Enhances Mycobacterium tuberculosis Control.

Early innate immune responses play an important role in determining the protective outcome of Mycobacterium tuberculosis (Mtb) infection. Nuclear factor κB (NF-κB) signaling in immune cells regulates the expression of key downstream effector molecules that mount early antimycobacterial responses. Using conditional knockout mice, we studied the effect of abrogation of NF-κB signaling in different myeloid cell types and its impact on Mtb infection. Our results show that the absence of IKK2-mediated signaling in all myeloid cells resulted in increased susceptibility to Mtb infection. In contrast, the absence of IKK2-mediated signaling in CD11c+ myeloid cells induced early proinflammatory cytokine responses, enhanced the recruitment of myeloid cells, and mediated early resistance to Mtb. Abrogation of IKK2 in MRP8-expressing neutrophils did not affect disease pathology or Mtb control. Thus, we describe an early immunoregulatory role for NF-κB signaling in CD11c-expressing phagocytes and a later protective role for NF-κB in LysM-expressing cells during Mtb infection.

CD11c+ macrophages are proangiogenic and necessary for experimental choroidal neovascularization.

Patients with neovascular AMD (nAMD) suffer vision loss from destructive angiogenesis, termed choroidal neovascularization (CNV). Macrophages are found in CNV lesions from patients with nAMD. Additionally, Ccr2-/- mice, which lack classical monocyte-derived macrophages, show reduced CNV size. However, macrophages are highly diverse cells that can perform multiple functions. We performed single-cell RNA-Seq on immune cells from WT and Ccr2-/- eyes to uncover macrophage heterogeneity during the laser-induced CNV mouse model of nAMD. We identified 12 macrophage clusters, including Spp1+ macrophages. Spp1+ macrophages were enriched from WT lasered eyes and expressed a proangiogenic transcriptome via multiple pathways, including vascular endothelial growth factor signaling, endothelial cell sprouting, cytokine signaling, and fibrosis. Additionally, Spp1+ macrophages expressed the marker CD11c, and CD11c+ macrophages were increased by laser and present in CNV lesions. Finally, CD11c+ macrophage depletion reduced CNV size by 40%. These findings broaden our understanding of ocular macrophage heterogeneity and implicate CD11c+ macrophages as potential therapeutic targets for treatment-resistant patients with nAMD.

Post-endotoxin exposure-induced lung inflammation and resolution consequences beneficially impacted by lung-delivered IL-10 therapy.

Although lung diseases typically result from long-term exposures, even a robust, one-time exposure can result in long-lasting consequences. Endotoxin is a ubiquitous environmental/occupational inflammatory agent often used to model airway inflammation. Using a murine model, the return to lung homeostasis following high dose inhalant lipopolysaccharide (LPS, 10-100 µg) exposure were delineated over 2 weeks. LPS-induced rapid weight loss, release of proinflammatory mediators, and inflammatory cell influx with prolonged persistence of activated macrophages CD11c+CD11b+ and recruited/transitioning CD11cintCD11b+ monocyte-macrophages out to 2 weeks. Next, lung-delivered recombinant (r) interleukin (IL)-10 was intratracheally administered for 3 doses initiated 5 h following LPS (10 µg) exposure for 2 days. IL-10 therapy reduced LPS-induced weight loss and increased blood glucose levels. Whereas there was no difference in LPS-induced bronchoalveolar lavage airway fluid cellular influx, total lung cell infiltrates were reduced (37%) with rIL-10 treatment. Post-LPS exposure treatment with rIL-10 strikingly reduced lavage fluid and lung homogenate levels of tumor necrosis factor-α (88% and 93% reduction, respectively), IL-6 (98% and 94% reduction), CXCL1 (66% and 75% reduction), and CXCL2 (47% and 67% reduction). LPS-induced recruited monocyte-macrophages (CD11cintCD11b+) were reduced (68%) with rIL-10. Correspondingly, LPS-induced lung tissue CCR2+ inflammatory monocyte-macrophage were reduced with rIL-10. There were also reductions in LPS-induced lung neutrophils, lymphocyte subpopulations, collagen content, and vimentin expression. These findings support the importance of studying resolution processes for the development of treatment after unintended environmental/occupational biohazard exposures. Short-term, lung-delivered rIL-10 favorably hastened inflammatory recovery processes following acute, high dose inhalant LPS exposure.

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice.

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (>10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells (DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

The Effect of Age and Cell Culture Parameters on the Quantity and Function of Bone Marrow-derived Dendritic Cells.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Conditional knockout of oestrogen receptor alpha in CD11c+ cells impacts female survival and inflammatory cytokine profile in murine lupus.

Oestrogen and oestrogen receptor alpha (ERα) have been implicated in systemic lupus erythematosus pathogenesis. ERα signalling influences dendritic cell (DC) development and function, as well as inflammation and downstream immune responses. We previously reported that ERα modulates multiple Toll-like receptor-stimulated pathways in both conventional and plasmacytoid DCs in lupus-prone mice. For example, CD11chi MHCII+ cell numbers are reduced in mice with global ERα deficiency or when expressing a short variant of ERα. Herein, RNA-seq analysis of CD11chi cells from bone marrow of NZM2410 mice expressing WT ERα versus ERα short versus ERα null revealed differentially expressed complement genes, interferon-related genes and cytokine signalling (e.g., IL-17 and Th17 pathways). To better understand the role of ERα in CD11c+ cells, lupus prone NZM2410 mice with selective deletion of the Esr1 gene in CD11c+ cells were generated. Phenotype and survival of these mice were similar with the exception of Cre positive (CrePos) female mice. CrePos females, but not males, all died unexpectedly prior to 35 weeks. DC subsets were not significantly different between groups. Since ERα is necessary for robust development of DCs, this result suggests that DC fate was determined prior to CD11c expression and subsequent ERα deletion (i.e., proximally in DC ontogeny). Overall, findings point to a clear functional role for ERα in regulating cytokine signalling and inflammation, suggesting that further study into ERα-mediated regulatory mechanisms in DCs and other immune cell types is warranted.

T-bet+ B cells Dominate the Peritoneal Cavity B Cell Response during Murine Intracellular Bacterial Infection.

T-bet+ B cells have emerged as a major B cell subset associated with both protective immunity and immunopathogenesis. T-bet is a transcription factor associated with the type I adaptive immune response to intracellular pathogens, driving an effector program characterized by the production of IFN-γ. Murine infection with the intracellular bacterium, Ehrlichia muris, generates protective extrafollicular T cell-independent T-bet+ IgM-secreting plasmablasts, as well as T-bet+ IgM memory cells. Although T-bet is a signature transcription factor for this subset, it is dispensable for splenic CD11c+ memory B cell development, but not for class switching to IgG2c. In addition to the T-bet+ plasmablasts found in the spleen, we show that Ab-secreting cells can also be found within the mouse peritoneal cavity; these cells, as well as their CD138- counterparts, also expressed T-bet. A large fraction of the T-bet+ peritoneal B cells detected during early infection were highly proliferative and expressed CXCR3 and CD11b, but, unlike in the spleen, they did not express CD11c. T-bet+ CD11b+ memory B cells were the dominant B cell population in the peritoneal cavity at 30 d postinfection, and although they expressed high levels of T-bet, they did not require B cell-intrinsic T-bet expression for their generation. Our data uncover a niche for T-bet+ B cells within the peritoneal cavity during intracellular bacterial infection, and they identify this site as a reservoir for T-bet+ B cell memory.

Human T-bet governs the generation of a distinct subset of CD11chighCD21low B cells.

High-level expression of the transcription factor T-bet characterizes a phenotypically distinct murine B cell population known as "age-associated B cells" (ABCs). T-bet-deficient mice have reduced ABCs and impaired humoral immunity. We describe a patient with inherited T-bet deficiency and largely normal humoral immunity including intact somatic hypermutation, affinity maturation and memory B cell formation in vivo, and B cell differentiation into Ig-producing plasmablasts in vitro. Nevertheless, the patient exhibited skewed class switching to IgG1, IgG4, and IgE, along with reduced IgG2, both in vivo and in vitro. Moreover, T-bet was required for the in vivo and in vitro development of a distinct subset of human B cells characterized by reduced expression of CD21 and the concomitantly high expression of CD19, CD20, CD11c, FCRL5, and T-bet, a phenotype that shares many features with murine ABCs. Mechanistically, human T-bet governed CD21loCD11chi B cell differentiation by controlling the chromatin accessibility of lineage-defining genes in these cells: FAS, IL21R, SEC61B, DUSP4, DAPP1, SOX5, CD79B, and CXCR4. Thus, human T-bet is largely redundant for long-lived protective humoral immunity but is essential for the development of a distinct subset of human CD11chiCD21lo B cells.

CD5L attenuates allergic airway inflammation by expanding CD11chigh alveolar macrophages and inhibiting NLRP3 inflammasome activation via HDAC2.

Allergic asthma is an airway inflammatory disease dominated by type 2 immune responses and there is currently no curative therapy for asthma. CD5-like antigen (CD5L) has been known to be involved in a variety of inflammatory diseases. However, the role of CD5L in allergic asthma remains unclear. In the present study, mice were treated with recombinant CD5L (rCD5L) during house dust mite (HDM) and ovalbumin (OVA) challenge to determine the role of CD5L in allergic asthma, and the underlying mechanism was further explored. Compared with PBS group, serum CD5L levels of asthmatic mice were significantly decreased, and the levels of CD5L in lung tissues and bronchoalveolar lavage fluid (BALF) were significantly increased. CD5L reduced airway inflammation and Th2 immune responses in asthmatic mice. CD5L exerted its anti-inflammatory function by increasing CD11chigh alveolar macrophages (CD11chigh AMs), and the anti-inflammatory role of CD11chigh AMs in allergic asthma was confirmed by CD11chigh AMs depletion and transfer assays. In addition, CD5L increased the CD5L+ macrophages and inhibited NLRP3 inflammasome activation by increasing HDAC2 expression in lung tissues of asthmatic mice. Hence, our study implicates that CD5L has potential usefulness for asthma treatment.

Ly6D+Siglec-H+ precursors contribute to conventional dendritic cells via a Zbtb46+Ly6D+ intermediary stage.

Plasmacytoid and conventional dendritic cells (pDC and cDC) are generated from progenitor cells in the bone marrow and commitment to pDCs or cDC subtypes may occur in earlier and later progenitor stages. Cells within the CD11c+MHCII-/loSiglec-H+CCR9lo DC precursor fraction of the mouse bone marrow generate both pDCs and cDCs. Here we investigate the heterogeneity and commitment of subsets in this compartment by single-cell transcriptomics and high-dimensional flow cytometry combined with cell fate analysis: Within the CD11c+MHCII-/loSiglec-H+CCR9lo DC precursor pool cells expressing high levels of Ly6D and lacking expression of transcription factor Zbtb46 contain CCR9loB220hi immediate pDC precursors and CCR9loB220lo (lo-lo) cells which still generate pDCs and cDCs in vitro and in vivo under steady state conditions. cDC-primed cells within the Ly6DhiZbtb46- lo-lo precursors rapidly upregulate Zbtb46 and pass through a Zbtb46+Ly6D+ intermediate stage before acquiring cDC phenotype after cell division. Type I IFN stimulation limits cDC and promotes pDC output from this precursor fraction by arresting cDC-primed cells in the Zbtb46+Ly6D+ stage preventing their expansion and differentiation into cDCs. Modulation of pDC versus cDC output from precursors by external factors may allow for adaptation of DC subset composition at later differentiation stages.