Loss of Cxcr4 in Endometriosis Reduces Proliferation and Lesion Number while Increasing Intraepithelial Lymphocyte Infiltration

Hyperactivation of the CXCL12-CXCR4 axis occurs in endometriosis; the therapeutic potential of treatments aimed at global inhibition of the axis was recently reported. Because CXCR4 is predominantly expressed on epithelial cells in the uterus, this study explored the effects of targeted disruption of CXCR4 in endometriosis lesions. Uteri derived from adult female mice homozygous for a floxed allele of CXCR4 and co-expressing Cre recombinase under control of progesterone receptor promoter were sutured onto the peritoneum of cycling host mice expressing the green fluorescent protein. Four weeks after endometriosis induction, significantly lower number of lesions developed in Cxcr4-conditional knockout lesions relative to those in controls (37.5% vs. 68.8%, respectively). In lesions that developed in Cxcr4-knockout, reduced epithelial proliferation was associated with a lower ratio of epithelial to total lesion area compared with controls. Furthermore, while CD3⁺ lymphocytes were largely excluded from the epithelial compartment in control lesions, in Cxcr4-knockout lesions, CD3⁺ lymphocytes infiltrated the Cxcr4-deficient epithelium in the diestrus and proestrus stages. Current data demonstrate that local CXCR4 expression is necessary for proliferation of the epithelial compartment of endometriosis lesions, that its downregulation compromises lesion numbers, and suggest a role for epithelial CXCR4 in lesion immune evasion.

Endometriosis is one of the most common gynecologic diseases in women of reproductive age, with a prevalence rate of approximately 10%.¹ Various theories have been proposed for the origin of endometriosis, including the most widely accepted theory of retrograde menstruation, in which shed endometrial tissue is refluxed through the fallopian tubes and proliferates within the pelvis.^2,3 Because the majority of women have retrograde menstruation, yet only about one in 10 develops endometriosis, it has been proposed that factors promoting survival, invasiveness, and growth of endometrial fragments in the peritoneal cavity play a role in their persistence at ectopic sites in women with endometriosis. Such predisposing factors include somatic mutations in the highly proliferative endometrial epithelium (ie, KRAS, ARID1A⁴), aberrant progenitor/stem cell populations (endometrial or bone marrow (BM) derived5, 6, 7, 8, 9) at ectopic sites, and/or an immune-tolerant microenvironment permissive to proliferation and neoangiogenesis of ectopic endometrial fragments. This immunosuppressive microenvironment is characterized by elevated levels of activated peritoneal macrophages, reduced natural killer cell activity, and abnormally high levels of T-regulatory cells,¹⁰ which suppress immune mechanisms aimed at eliminating implantation of misplaced autologous cells.The chemokine-receptor CXCL12-CXCR4 axis is up-regulated in endometriosis.11, 12, 13, 14 The axis has roles in promoting cell survival, proliferation, chemotaxis, invasion, and angiogenesis. In cancer, hyperactivation of the axis is associated with disease progression and correlates with poor clinical outcome.15, 16, 17, 18, 19 This axis was also proposed to function in immune modulation: CXCL12 binding to CXCR4-expressing intratumoral (epithelial) cells was suggested to be a mechanism mediating cancer evasion of immune surveillance.^20,21 Therapeutic blockade of the axis with the CXCR4 antagonist AMD3100 exhibited antitumor effects, including reduced tumor proliferation and increased apoptosis, both associated with T-cell accumulation within the tumor epithelium.^20,22, 23, 24Endometrial CXCR4 is predominantly expressed on luminal and glandular epithelia, whereas the stroma is the principal source of the ligand CXCL12.^13,25 Stromal-derived CXCL12 exerts its proliferative effect on the epithelium through paracrine interactions with its cognate receptor CXCR4.²⁶ Estradiol stimulates CXCL12 production and progesterone to inhibit this stimulation.^27,28 In vitro, AMD3100 blocked the CXCL12-mediated proliferative effects on epithelial cells.²⁹ Acute treatment of experimental endometriosis in mice with AMD3100 significantly decreases lesion volume and reduces BM cell trafficking to lesions.³⁰ AMD3100 was also shown to reduce recruitment of BM-derived endothelial progenitor cells into lesions and compromise their vascularization.³¹ Based on these studies, whether the inhibitory action of AMD3100 on lesion growth is mediated via local effects (ie, inhibiting lesion-endogenous CXCR4) or systemic effects (ie, inhibiting exogenous CXCR4-expressing cells, which infiltrate lesions with endometriosis induction) was explored. Moreover, in a manner similar to cancer, lesion-derived CXCR4 may have a role in immune evasion.To achieve these goals, endometriosis was induced using uteri derived from 8- to 10- week–old Pgr^Cre/+ Cxcr4^−/− female mice homozygous for a floxed CXCR4 allele and harboring a progesterone receptor promoter–driven Cre recombinase. Endometriosis was induced in syngeneic green fluorescent protein (GFP) transgenic host mice, allowing discrimination of host-derived populations from endometrial cells within uterine explants. A significant reduction in the number of lesions was found in mice harboring Cxcr4-conditional knockout lesions. In lesions that did develop, epithelial thinning was observed concomitant with the appearance of intraepithelial lymphocytes. At the proliferative stage, Ki-67 staining was absent from the epithelium of lesions, suggesting that diminished lesion numbers may be attributed to loss of epithelial proliferation, ultimately undermining lesion integrity.     

A Novel Mouse Model of Endometriosis Mimics Human Phenotype and Reveals Insights into the Inflammatory Contribution of Shed Endometrium

Endometriosis is an estrogen-dependent inflammatory disorder characterized by the presence of endometrial tissue outside the uterine cavity. Patients experience chronic pelvic pain and infertility, with the most likely origin of the tissue deposits (lesions) being endometrial fragments shed at menses. Menstruation is an inflammatory process associated with a dramatic increase in inflammatory mediators and tissue-resident immune cells. In the present study, we developed and validated a mouse model of endometriosis using syngeneic menstrual endometrial tissue introduced into the peritoneum of immunocompetent mice. We demonstrate the establishment of endometriotic lesions that exhibit similarities to those recovered from patients undergoing laparoscopy. Specifically, in both cases, lesions had epithelial (cytokeratin⁺) and stromal (vimentin/CD10⁺) cell compartments with a well-developed vasculature (CD31⁺ endothelial cells). Expression of estrogen receptor β was increased in lesions compared with the peritoneum or eutopic endometrium. By performing experiments using mice with green fluorescent protein–labeled macrophages (MacGreen) in reciprocal transfers with wild-type mice, we obtained evidence that macrophages present in the peritoneum and in menses endometrium can contribute to the inflammatory microenvironment of the lesions. In summary, we developed a mouse model of endometriosis that exhibits similarities to human peritoneal lesions with respect to estrogen receptor expression, inflammation, and macrophage infiltration, providing an opportunity for further studies and the possible identification of novel therapies for this perplexing disorder.Endometriosis occurs in 6% to 10% of women of reproductive age,¹ is associated with chronic pelvic pain and infertility, and represents a huge socioeconomic burden.² The defining feature of the condition is the presence of endometrial tissue outside the uterine cavity, typically on the peritoneal wall³ or the surface of the ovary (endometriomas).⁴ Spontaneous endometriosis occurs only in humans and some primates,^5,6 where the luminal portion of the endometrium sheds at the end of the menstrual cycle. The primary cause of endometriosis is widely accepted to be the introduction of endometrial tissue into the peritoneal cavity via the fallopian tubes, a process known as retrograde menstruation.⁷ In captive baboons, the incidence of spontaneous endometriosis parallels the number of menstrual cycles experienced, providing a link between shedding of endometrial tissue and the onset of disease.⁸Current treatment strategies for peritoneal endometriosis are restricted to surgical excision of the lesions or suppression of ovarian function to mimic premature menopause. In up to 75% of cases, symptoms recur after surgery, with long-term ovarian suppression sometimes ineffective.⁹ Although it is acknowledged that there is an unmet clinical need for new nonsurgical treatments for endometriosis, their development depends on access to animal models that can recapitulate features of the human disorder.¹⁰ Nonhuman primates offer a physiologically relevant model of endometriosis, although their use is limited by cost and ethical concerns. Injecting female baboons with autologous menstrual endometrium into the peritoneal cavity has been shown to result in the development of endometriotic lesions.¹¹ This model has been used in longitudinal studies, allowing new insights into changes in gene expression in lesions as they become established.¹² In studies using the same model, expression of aromatase and the β isoform of estrogen receptor (ERβ) was found to be increased in the lesions, a finding in agreement with studies in women.^13,14 Rat and mouse models of endometriosis have also been developed, offering the advantage of lower cost and smaller animal size for testing potential therapies. The availability of mice genetically engineered to express fluorescent marker proteins^15,16 or targeted deletion of genes implicated in endometrial disease progression¹⁷ has made them an attractive alternative to primate studies. To date, studies using mice have usually involved the injection of human endometrial tissue into immunosuppressed mice¹⁸ or the introduction of small fragments of mouse uterine tissue that are sutured to the peritoneum or other sites (reviewed in Grummer¹⁹). Results obtained using both models have provided new insights into the origins of the disorder and its association with infertility²⁰ and have served as a platform for testing potential therapies, including those targeting angiogenesis in the lesions²¹; however, both models have limitations. For example, although human tissue injected either under the skin or into the peritoneal cavity can result in the formation of endometriosis-like lesions, these can grow only if the recipients lack the capacity to mount a tissue rejection response, eg, they are homozygous for the Prkdc^SCID mutation.¹⁸ Models using mice with an intact immune system allow for studies on the role(s) of inflammatory cytokines and immune cells,^22,23 but because mice do not exhibit spontaneous decidualization or menstruation, the uterine tissue introduced into the peritoneal cavity does not recapitulate the tissue microenvironment at the time of retrograde menstruation. Furthermore, in most of the autologous/syngeneic mouse models, uterine tissue is secured in the cavity after local abrasion and/or using sutures,^16,22 with both procedures likely to induce an artificial inflammatory response.In the present study, we set out to develop a mouse model of endometriosis that would combine the best features of the current baboon and mouse models. For this we used our recently validated mouse model of menstruation²⁴ as the source of syngeneic mouse menstrual endometrium and introduced this into the peritoneum of immunocompetent mice. To determine whether this approach could mimic the estrogenic and inflammatory features of human peritoneal lesions, we examined gene and protein expression in mice and patients and complemented these studies by undertaking reciprocal transfers between mice in which cells of the granulocyte/macrophage lineage were labeled with green fluorescent protein (GFP).¹⁵     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.A large body of evidence indicates that soluble oligomers of amyloid-β (Aβ) are the primary toxic peptides that initiate downstream tau pathology in the amyloid cascade hypothesis of Alzheimer disease (AD).^{1, 2} However, the time course and severity of AD dementia have been generally found to correlate with neurofibrillary tangle development rather than plaque appearance,^{3, 4, 5, 6, 7, 8} although a few studies have linked plaques with early cognitive decline.^{9, 10, 11, 12} Soluble oligomeric Aβ has been highlighted as the primary toxin for loss of dendritic spines and synaptic function¹³ and has also been directly linked to downstream tau pathology. For example, suppression of a tau kinase pathway can prevent Aβ42 oligomer-induced dendritic spine loss,¹⁴ and injection of Aβ42 fibrils into mutant tau mice induces neurofibrillary tangles in cell bodies retrograde to the injections.¹⁵ In vivo, effects of Aβ oligomers versus fibrils are harder to separate; however, lowering soluble Aβ oligomers by halving β–site amyloid precursor protein (APP) cleaving enzyme reduces accumulation and phosphorylation of wild-type tau in a mouse model.¹⁶ Evidence for Aβ and tau association is particularly strong in the dendritic compartment, where tau was shown to mediate Aβ toxicity via linkage of fyn to downstream N-methyl-d-aspartate receptor toxicity.¹⁷The earliest cognitive losses in AD have long been thought to correlate with synapse loss.^{8, 18, 19, 20, 21} In humans, electron microscopic studies have documented synapse-associated Aβ and tau,^{22, 23} and much work documents activity-dependent release of synaptic Aβ into interstitial fluid, which drives local Aβ deposition in human subjects and in rodents.^{4, 24, 25} Of importance, most synapse-associated Aβ in cortical synapses of AD patients consists of soluble oligomeric species,²⁶ and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers.^{27, 28, 29, 30, 31} With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Aβ and hyperphosphorylated tau (p-tau) pathology in synaptic terminals. In addition to early- and late-stage disease, the AD samples included nondemented high pathology controls (HPCs) with substantial AD-related pathology. Synaptic accumulation of Aβ occurred in the earliest plaque stages, before the appearance of synaptic p-tau, which did not appear until late-stage disease. Soluble Aβ oligomers in synaptic terminals were elevated in early AD cases compared with HPCs, indicating an association with the onset of a dementia diagnosis.     

Altered Macrophage Phenotype Transition Impairs Skeletal Muscle Regeneration

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11b-diphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b⁺(CD90/B220/CD49b/NK1.1/Ly6G)⁻(F4/80/I-Ab/CD11c)⁻Ly6C^+/−] accumulation occurred; interestingly, Ly6C⁺ and Ly6C⁻ monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C⁺ macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b⁺F4/80⁺ cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b⁺F4/80⁺Ly6C⁻CD301⁻ macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of anti-inflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C⁻ macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration.Skeletal muscle has a remarkable capacity for regeneration with a complex injury/repair process that includes inflammation, myofiber regeneration, and angiogenesis. The careful orchestration of inflammatory cells and resident muscle stem cells, also known as satellite cells, is vital to skeletal muscle regenerative capacity.^1,2Macrophages are unique effector cells in innate immunity that play critical roles in the maintenance of tissue homeostasis. Monocytes/macrophages are major inflammatory cell populations recruited into injured skeletal muscle. Mouse monocytes comprise two phenotypically distinct subsets in blood: Ly6C^hi/+ cells and Ly6C^lo/− cells.^3–5 Although some groups suggest that Ly6C^hi/+ monocytes are solely recruited into injured tissue and further become Ly6C^lo/− monocytes within the tissue,^3,4,6,7 others have suggested that Ly6C^lo/− monocytes can also be recruited as a second wave to the injury site after the immediate response by Ly6C^hi/+ cells.^8,9 Swirski et al⁵ further characterized and expanded the definition of Ly6C^hi and Ly6C^lo monocyte subsets in the blood [specifically, CD11b⁺(F480, I-Ab, CD11c)^lo(CD90, B220, CD49b, NK1.1, Ly6G)^loLy6C^hi/lo], which were shown to be derived from splenic reserves and recruited to injured myocardium following a myocardial infarction. However, the propensity of Ly6C^hi/+ and Ly6C^lo/− monocytes to differentiate into specific macrophage polarization states (ie, M1 or M2) has not been established. Ramachandran et al⁶ provided evidence that Ly6C^hi monocytes were recruited to injured liver and further differentiated into both Ly6C^hi and Ly6C^lo macrophages. Interestingly, many groups have characterized the Ly6C^hi/+ monocytes/macrophages as proinflammatory, primarily by their production/expression of proinflammatory cytokines/chemokines such as chemokine ligand-2 [Ccl2, also known as monocyte chemoattractant protein-1 (Mcp-1)],^5,6 Cxcl10 [interferon-γ–induced protein 10 (Ip-10)],⁶ inducible nitric oxide synthase (iNOS),¹⁰ tumor necrosis factor-α (TNF-α)^7,8,10,11, IL-12,¹⁰ Cxcl2 [macrophage inflammatory protein-2 (Mip-2)],³ Il-1β,^3,7 and vascular endothelial growth factor (VEGF).⁵ These groups have also characterized Ly6C^lo/− monocytes/macrophages as anti-inflammatory by their production/expression of anti-inflammatory cytokine/chemokines, growth factors, or other markers such as Ccl22 (Mdc),³ Ccl17 (Tarc),³ Il-4,⁵ IL-10,^5,7 transforming growth factor-β (Tgf-β),⁷ arginase-1 (Arg1),^6,12 resistin-like alpha [Retnla, also known as found in inflammatory zone-1 (Fizz-1)],⁶ insulin-like growth factor-1 (Igf-1),^3,6 and platelet-derived growth factor-β (Pdgf-β).³ Whereas Ly6C⁺ cells have a short half-life during tissue damage,⁴ Ly6C⁻ cells remain in the circulation for longer periods and traffic into peripheral tissues under noninflammatory conditions.^4,13 The dynamics of Ly6C^hi/+ and Ly6C^lo/− monocyte infiltration into injured skeletal muscle and their propensity to contribute to proinflammatory and anti-inflammatory macrophages in this tissue remain to be fully explored.Although monocytes are recruited to injured tissues, resident macrophages also exist in tissue and play a role in inflammation. Brigitte et al¹⁴ found that resident macrophages form a centripetal migration pathway for recruited leukocytes by producing two chemokines, KC and MCP-1. Two studies selectively ablated resident macrophages using the human diphtheria toxin receptor (DTR) present on CD11b-expressing cells (ie, monocytes/macrophages/neutrophils). One study showed that the intraperitoneal injection of diphtheria toxin (DT) in a CD11b-DTR mouse could ablate resident macrophages in the kidney and ovary, but not the hepatic sinusoidal nor alveolar macrophages.¹⁵ By using a chimeric mouse, CD11b-DTR host with green fluorescent protein (GFP) donor bone marrow (BM), another group¹⁴ demonstrated a reduction in the resident macrophage population in skeletal muscle with intravenous DT treatment. Consequently, a reduction in the recruited GFP⁺ population 1 day after muscle injury compared to control was observed.¹⁴ However, as the transplanted BM was GFP⁺, it is unclear whether this recruitment deficit was present in the neutrophil population (the primary myeloid cell recruited at day 1), whether this affected macrophage recruitment at any time points along muscle regeneration, or whether this had any effect on the regeneration of the muscle. Regardless, the essential role of ablation of resident macrophages in skeletal muscle regeneration remains elusive.The concept that macrophages are crucial in muscle regeneration is supported by growing experimental evidence. Firstly, several different methods have been used to deplete monocytes/macrophages to investigate their role in skeletal muscle regeneration. This includes injecting antibodies against F4/80,¹⁶ CD11b,^17,18 macrophage colony-stimulating factor (M-CSF) receptor,¹⁹ or clodronate-containing liposomes¹; all of these experiments demonstrated that monocyte/macrophage depletion impaired skeletal muscle regeneration. Secondly, mice deficient in Mcp-1 (also known as Ccl2) or the MCP-1 receptor, CC chemokine receptor 2 (Ccr2), demonstrated a remarkable decrease in monocyte/macrophage infiltration in association with impaired skeletal muscle regeneration.^20–24 Most importantly, the poor capacity of skeletal muscle regeneration in Ccr2^−/− mice was restored by transplantation with BM-derived cells from wild-type mice.²⁵ Thus, Ccr2 expression by BM-derived cells is critical in skeletal muscle regeneration, an observation that strongly supports the essential role for monocytes/macrophages in this dynamic process.Macrophages exhibit remarkable plasticity and are physiologically diverse in response to environmental cues. Extensive in vitro studies defined two phenotypically different macrophage subsets by activation state.^26–29 Classically activated, or M1 macrophages, obtained by lipopolysaccharide treatment alone or in combination with IFN-γ, secrete proinflammatory cytokines, such as TNF-α, and increased iNOS activity, resulting in the production of reactive oxygen species. Alternatively activated, or M2 macrophages, are activated by IL-4 treatment. M2 macrophages express high Arg1, Ym1, Fizz1,¹² the mannose receptor (CD206), and CD301.^2,30,31 Multiple variants based on different stimuli have been described and designated as M2a, M2b, and M2c,^26,27 or as wound healing and regulatory macrophages.²⁸Monocyte and macrophage subsets also exist in injured skeletal muscle. Following injury, proinflammatory and anti-inflammatory monocytes/macrophages sequentially accumulated in muscle. Initial monocyte/macrophage populations were associated with the production of proinflammatory cytokines and removal of necrotic tissue. These initial populations were replaced by monocytes/macrophages that were associated with the production of anti-inflammatory cytokines and tissue repair.^7,32 In rats, M1 and M2 subsets were defined as ED1⁺ and ED2⁺ macrophages, respectively.^33,34 Although these studies suggest that different monocyte/macrophage subsets are associated with different stages of skeletal muscle regeneration, the kinetics and influence of different monocyte/macrophage subsets in skeletal muscle regeneration remain to be determined.DTR transgenic mice have been used to study the effects of monocyte/macrophage ablation on tissue injury and repair.^7,15,35,36 DT binds to the heparin-binding epidermal growth factor–like growth factor (hbEGF) receptor (also known as DTR) followed by internalization, rapidly inducing apoptosis in both dividing and terminally differentiated cells.³⁷ DT exhibits 1 × 10⁴ less affinity in normal mouse cells compared to human cells.³⁸ Thus, tissue-specific transgenic expression of human hbEGF (DTR) confers DT sensitivity to murine cells, such as dendritic cells, vascular smooth muscle cells, or monocytes/macrophages.^15,39,40 CD11b-DTR mice express a transgene containing a human DTR under the control of the CD11b promoter that is constitutively expressed in monocytes and macrophages. Ablation studies in peritoneal populations revealed specific ablation of F4/80⁺ populations in CD11b-DTR mice that were not affected when DT was injected into wild-type mice. Additionally, other population cell numbers such as of T cells, B cells, and granulocytes in the spleen and peritoneal cavity were not affected by DT administration in CD11b-DTR mice.⁴¹ Therefore, monocytes/macrophages can be transiently and specifically ablated by treatment with a single dose of DT.In this study, we used DTR transgenic mice on a FVB background (CD11b-DTR) to transiently ablate monocytes/macrophages at different time points following injury to investigate the effect of monocyte and macrophage subsets on skeletal muscle regeneration. Ablating early infiltrating monocytes/macrophages impaired skeletal muscle regeneration, whereas later ablation had a minimal effect on this tissue response to injury.     

The Impact of IFN-γ Receptor on SLPI Expression in Active Tuberculosis: Association with Disease Severity

Interferon (IFN)-γ displays a critical role in tuberculosis (TB), modulating the innate and adaptive immune responses. Previously, we reported that secretory leukocyte protease inhibitor (SLPI) is a pattern recognition receptor with anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb). Herein, we determined whether IFN-γ modulated the levels of SLPI in TB patients. Plasma levels of SLPI and IFN-γ were studied in healthy donors (HDs) and TB patients. Peripheral blood mononuclear cells from HDs and patients with TB or defective IFN-γ receptor 1* were stimulated with Mtb antigen and SLPI, and IFN-γR expression levels were measured. Both SLPI and IFN-γ were significantly enhanced in plasma from those with TB compared with HDs. A direct association between SLPI levels and the severity of TB was detected. In addition, Mtb antigen stimulation decreased the SLPI produced by peripheral blood mononuclear cells from HDs, but not from TB or IFN-γR patients. Neutralization of IFN-γ reversed the inhibition of SLPI induced by Mtb antigen in HDs, but not in TB patients. Furthermore, recombinant IFN-γ was unable to modify the expression of SLPI in TB patients. Finally, IFN-γR expression was lower in TB compared with HD peripheral blood mononuclear cells. These results show that Mtb-induced IFN-γ down-modulated SLPI levels by signaling through the IFN-γR in HDs. This inhibitory mechanism was not observed in TB, probably because of the low expression of IFN-γR detected in these individuals.Tuberculosis (TB) is among the most common causes of morbidity and mortality in patients with HIV infection. Although protective immunological mechanisms against Mycobacterium tuberculosis (Mtb) are not fully understood, resistance to mycobacterial infections is primarily mediated by the interaction of antigen-specific T cells and macrophages.^1,2 This interaction depends on the cross talk of cytokines produced by these cells, and interferon (IFN)-γ is essential for protection.^2,3 Thus, during the immune response of the host against Mtb, IFN-γ produced by type 1 helper T cells is recognized by its receptor on macrophages. The IFN-γ receptor (IFN-γR) is composed of two ligand-binding IFNGR1 chains associated with two signal-transducing IFNGR2 chains, and an associated signaling machinery.^2–5 IFN-γ binds to its receptor and activates macrophages to efficient killing of intracellular mycobacteria. In humans, the loss-of-function mutations in IFNGR1 or IFNGR2 genes are closely associated with severe susceptibility to poorly virulent mycobacteria highlighted in childhood.^4,6,7Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor secreted by inflammatory and epithelial cells, mainly in the respiratory tract mucosa, and it is primarily active against neutrophilic elastase, cathepsin G, trypsin, and chymotrypsin.⁸ The expression and secretion of SLPI are down-modulated during chronic obstructive pulmonary disease.^9–11 In addition, cathepsins B, L, and S and cigarette smoke exposure result in the cleavage and inactivation of SLPI.^12,13 Moreover, it has been demonstrated that IFN-γ is a prominent stimulator of cathepsins and matrix metalloproteinase-12 and an inhibitor of SLPI.¹⁴ Remarkably, SLPI may also function as an endogenous immunomodulatory, anti-inflammatory, and/or antimicrobial substance.^15–18 The antimicrobial effects of SLPI against several bacteria have been demonstrated.¹⁵ In particular, Nishimura et al¹⁷ described that recombinant mouse SLPI inhibited the growth of bacillus Calmette-Guérin (BCG) and Mtb through the disruption of the mycobacterial cell wall structure. Furthermore, we reported that human SLPI is a secreted pattern recognition receptor for mycobacteria that increases both the phagocytosis and killing of the pathogen.¹⁸ Remarkably, exposure of murine peritoneal macrophages to Mtb led to an increase in SLPI secretion.¹⁹ Thus, given the anti-inflammatory and anti-mycobacterial roles of SLPI in humans and taking into account the fact that SLPI is inhibited by IFN-γ,²⁰ a crucial cytokine in the protective immunity against Mtb, herein we studied the effect of IFN-γ on the expression of SLPI during human active disease.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.Macrophages are versatile cells that have been long recognized as immune effectors where their recruitment to sites of injury is a fundamental feature of inflammation. Although their role in host defense has been well documented, macrophages and their precursors are also important during embryogenesis, normal tissue maintenance, and postnatal organ repair.^1,2 Almost all developing organs contain a population of resident monocytes that infiltrate very early during organogenesis and persist throughout adult life.^3–6 In addition to their phagocytic capabilities during tissue remodeling-associated apoptosis,^5,7 fetal macrophages have many trophic effects that promote tissue and organ growth.^6,8,9Colony-stimulating factor (CSF)-1 controls the differentiation, proliferation, and survival of macrophages by binding to a high-affinity cell-surface tyrosine kinase receptor (CSF-1R), encoded by the c-fms proto-oncogene that is expressed on macrophages and their progenitors.⁶ CSF-1 is critical for both adult and embryonic macrophage development. This is manifested by multiple organ growth deficiencies observed in osteopetrotic (Csf1^op/Csf1^op) mice that have a spontaneous mutation in the csf-1 gene. These mice show growth restriction and developmental abnormalities of the bones, brain, and reproductive and endocrine organs,^10–13 a phenotype that can be rescued by injection of exogenous CSF-1 or insertion of a csf-1 transgene.^14–16In adult organs, there is considerable heterogeneity of monocytes and macrophages with distinct subsets defined by phenotype, function, and the differential expression of cell surface markers.^17–19 Subpopulations of macrophages directly contribute to wound healing and tissue repair, supporting the concept that some macrophage phenotypes can promote organ regeneration after a pro-inflammatory state of injury.²⁰ The concept of macrophage polarization states has emerged; the M1 “classically activated” pro-inflammatory cell type apparently opposed by an M2 “alternatively activated” immune regulatory macrophage.¹⁸ In general, these two states are thought to be analogous to the opposing T helper 1 and T helper 2 immune responses, although in both cases this model is probably too simplistic. Functionally, it is more likely that distinct subpopulations of macrophages may exist in the same tissue and play critical roles in both the injury and recovery phases of inflammatory scarring.²⁰Our previous study provided evidence that the addition of CSF-1 to a developing murine kidney promotes a growth and differentiation response that is accompanied by increased numbers of macrophages.³ Furthermore, with the use of expression profiling we demonstrated that fetal kidney, lung, and brain macrophages share a characteristic gene expression profile that includes the production of factors important in the suppression of inflammation and the promotion of proliferation.³ Embryonic macrophages appear to play a positive trophic role that may have parallel reparative functions in many adult tissues undergoing repair and cellular replacement.^1,20 A number of studies have suggested that infiltrating macrophages along with the trophic factors they release participate in tissue repair of the kidney,^20–22 brain,²³ skin,^24,25 lung,²⁶ liver,²⁷ heart,²⁸ gastrointestinal tract,^29,30 and skeletal muscle.^31,32 Indeed, the pleiotrophic roles for CSF-1 in reproduction, development of multiple organ systems, and maternal-fetal interactions during pregnancy by macrophage-mediated processes have also been well defined.^2,33,34To determine the physiological relevance of CSF-1 as a component of the mammalian growth regulatory axis, CSF-1 was administered to neonatal mice. We report that CSF-1 administration to newborn mice increased body weight and kidney weight and volume and was associated with increased numbers of macrophages. Our results also establish that CSF-1 injection into mice after ischemia-reperfusion (IR) injury promoted endogenous repair with characteristic rapid re-epithelialization of the damaged tubular epithelium, leading to functional recovery. Flow cytometric and gene expression analyses were used to delineate the macrophage profile present in the kidneys during the early and resolution phase of IR injury with and without CSF-1 therapy. We thus provide evidence that CSF-1 recruits macrophages to the reparative site and influences their phenotype, partly through an insulin-like growth factor (IGF)-1 signaling response. Therefore, macrophages under the stimulus of CSF-1 in an acute setting of renal disease markedly accelerate renal cell replacement and tissue remodeling while attenuating downstream interstitial extracellular matrix accumulation.     

CXCL9 Is Important for Recruiting Immune T?Cells into the Brain and Inducing an Accumulation of the T Cells to the Areas of Tachyzoite Proliferation to Prevent Reactivation of Chronic Cerebral Infection with Toxoplasma gondii

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non–glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4⁺ and CD8⁺ T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3⁺ T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.Toxoplasma gondii is an obligate intracellular parasite in humans and animals. Interferon (IFN)-γ–mediated immune responses^{1, 2} and, to a lesser degree, humoral immunity^{3, 4, 5} control the tachyzoite growth during the acute stage of infection, but the parasite establishes a chronic infection by forming cysts preferentially in the brain. Chronic infection with T. gondii is ubiquitous in humans, and 500 million to 2 billion people worldwide are estimated to be chronically infected with this parasite.^{6, 7} This chronic infection can be reactivated and develop life-threatening toxoplasmic encephalitis (TE) in immunocompromised persons such as those with AIDS, neoplastic diseases, and organ transplants.^{8, 9} This fact clearly indicates an importance of the protective immunity to maintain the latency of chronic infection with T. gondii in the brain. However, the mechanisms by which the immune responses prevent reactivation of the chronic infection are not well understood.Although T. gondii has three major genotypes (types I, II, and III), type II is predominant in the strains isolated from patients with TE in North America and Europe.^{10, 11, 12} Therefore, for investigating the mechanisms by which the immune system maintains the latency of chronic T. gondii infection and prevents TE, animals that establish a latent, chronic infection with type II parasite in their brains provide an excellent model. BALB/c mice are one of those animals.^{13, 14} IFN-γ is essential to maintain the latency of chronic cerebral infection with T. gondii.^{15, 16} This cytokine can activate microglia^{17, 18} and astrocytes^{19, 20, 21} to prevent tachyzoite proliferation. In addition, IFN-γ plays an important role in regulating recruitment of immune T cells into the brain of BALB/c mice during both the acute and chronic stages of infection.^{22, 23} Induction of vascular cell adhesion molecule 1 (VCAM-1) expression on the cerebral vessels during the chronic infection is largely mediated by IFN-γ,²² and the binding of α4β1 integrin expressed on the surface of T cells to VCAM-1 expressed on the cerebrovascular endothelial cells is important for inducing prompt recruitment of immune T cells into their brains during the early stage of reactivation of chronic T. gondii infection to prevent TE.²⁴Chemokines, in addition to adhesion molecules, are crucial for T-cell entry into various organs.^{25, 26} In BALB/c mice chronically infected with T. gondii, CXCL9, CXCL10, and CCL5 are the chemokines predominantly expressed in their brains.^{27, 28} In the present study, we examined the role of IFN-γ in cerebral expression of these three chemokines during reactivation of the chronic infection in BALB/c-background immunodeficient mice, and found that the CXCL9 expression requires IFN-γ. On the basis of this observation, we examined the role of CXCL9 in recruiting immune T cells into the brain for maintaining the latency of chronic infection with T. gondii with the use of a model of reactivation of the infection in severe combined immunodeficient (SCID) mice with adoptive transfer of immune T cells from infected wild-type animals. By applying anti-CXCL9 antiserum in this animal model, the present study revealed that CXCL9 is crucial for recruiting immune T cells into the brain and for inducing an accumulation of the T cells around the areas associated with tachyzoites to prevent reactivation of cerebral infection with T. gondii.     

Pyroglutamate-3 Amyloid-β Deposition in the Brains of Humans,Non-Human Primates,Canines, and Alzheimer Disease–Like Transgenic Mouse Models

Amyloid-β (Aβ) peptides, starting with pyroglutamate at the third residue (pyroGlu-3 Aβ), are a major species deposited in the brain of Alzheimer disease (AD) patients. Recent studies suggest that this isoform shows higher toxicity and amyloidogenecity when compared to full-length Aβ peptides. Here, we report the first comprehensive and comparative IHC evaluation of pyroGlu-3 Aβ deposition in humans and animal models. PyroGlu-3 Aβ immunoreactivity (IR) is abundant in plaques and cerebral amyloid angiopathy of AD and Down syndrome patients, colocalizing with general Aβ IR. PyroGlu-3 Aβ is further present in two nontransgenic mammalian models of cerebral amyloidosis, Caribbean vervets, and beagle canines. In addition, pyroGlu-3 Aβ deposition was analyzed in 12 different AD-like transgenic mouse models. In contrast to humans, all transgenic models showed general Aβ deposition preceding pyroGlu-3 Aβ deposition. The findings varied greatly among the mouse models concerning age of onset and cortical brain region. In summary, pyroGlu-3 Aβ is a major species of β-amyloid deposited early in diffuse and focal plaques and cerebral amyloid angiopathy in humans and nonhuman primates, whereas it is deposited later in a subset of focal and vascular amyloid in AD-like transgenic mouse models. Given the proposed decisive role of pyroGlu-3 Aβ peptides for the development of human AD pathology, this study provides insights into the usage of animal models in AD studies.Alzheimer disease (AD) is the most common form of dementia, predicted to affect approximately 42 million people worldwide in the year 2020.¹ The two prominent histopathological hallmarks of AD are extracellular neuritic plaques composed of aggregated amyloid-β protein (Aβ) and intracellular neurofibrillary tangles comprising hyperphosphorylated tau.^2,3 Aβ is formed via the amyloidogenic pathway in which the amyloid precursor protein (APP) is liberated by two sequential endopeptidase cleavages (β- and γ-secretase).⁴ Besides a marked C-terminal heterogeneity of Aβ peptides represented by the isoforms Aβ40 and Aβ42, N-terminal variants are also frequently found, eg, pyroGlu-3 Aβ and pyroGlu-11 Aβ.⁵N-terminally truncated and modified Aβ species have been shown to be a major component of Aβ deposited in plaques and vessels of AD and Down syndrome (DS) patients.^6–9 Current hypotheses suggest that pyroGlu-3 Aβ may play an early and seminal role in the oligomerization and seeding of Aβ in familial AD (FAD) and sporadic AD.^10–12 PyroGlu-3 Aβ is formed by cyclization of glutamate residues 3 or 11 by glutaminyl cyclase (QC).¹³ An N-terminal truncation of Aβ precedes formation of pyroglutamic acid. Such post-translationally modified species have been shown to be highly toxic to neuronal and glial cultures.¹⁴ When compared to unmodified Aβ, pyroGlu-3 Aβ has a higher aggregation propensity and stability, and exhibits increased potential to interfere with hippocampal LTP.^15–17 Inhibition of QC has been shown to prevent pyroGlu-3 Aβ formation in vitro¹⁸ and in vivo.^19,20 Accordingly, QC inhibitors are currently in development as a novel pharmacological approach to treat and/or prevent AD.²⁰With the advent of numerous preclinical models, it is now possible to mimic at least some of the pathological hallmarks of AD for characterization and testing of potential treatments. With regard to the established role of pyroGlu-3 Aβ in AD pathology represented by neuron loss and cognitive decline,^12,21,22 a detailed characterization of pyroGlu-3 Aβ distribution of available transgenic (tg) and nontransgenic models and their comparison to human pathology is urgently needed. In this regard, recent research has focused only on select models.^23–26Here, we completed detailed IHC analyses in human AD, nondemented aged controls (AC), and DS brains, as well as in the Caribbean vervet,²⁷ aged beagle canines,²⁸ and 12 AD-like tg mouse strains. Our results support the early deposition of pyroGlu-3 Aβ along with general Aβ in humans, nonhuman primates, and canines, in contrast to later deposition in AD-like tg mouse models, underlining differences in plaque pathology between nontransgenic/nonrodent and transgenic murine model systems.     

p120-Catenin Expressed in Alveolar Type II Cells Is Essential for the Regulation of Lung Innate Immune Response

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell–specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to ¹²⁵I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.Lungs are constantly exposed to pathogens; therefore, a highly restrictive alveolar epithelial barrier and finely tuned host defense mechanisms are indispensable for their protection.^1,2 Unchecked inflammation is linked to various acute and chronic diseases, including edema, acute respiratory distress syndrome, and fibrosis.^3,4 Although it is abundantly clear that the alveolar epithelial barrier regulates the transport of gases, liquid, and ions,^5,6 the role of the barrier in the regulation of the innate immune function of lungs remains poorly understood.The restrictiveness of the alveolar epithelial barrier is dependent on a series of interacting proteins comprising the adherens junctions (AJs) and tight junctions (TJs).^7,8 The core of the epithelial AJs is composed of E-cadherin, which links cells to one another in the monolayer.⁹ The cytoplasmic domain of E-cadherin associates with α-catenin, β-catenin, and p120-catenin (p120, official name catenin delta 1; CTNND1).⁹ The α- and β-catenins can recruit proteins that link E-cadherin to the actin cytoskeleton,⁹ and together, these interactions maintain the tension landscape in the epithelial monolayer.¹⁰ β-Catenin also plays an essential role in the Wnt signaling pathway and thereby contributes to cell proliferation and differentiation.¹¹ However, p120 has received comparatively less attention, although recent studies have shown that p120 has important functions in regulating cadherin stability and turnover¹² and innate immunity.¹³Here, we focused on the role of p120 expressed in alveolar epithelial type II cells in regulating the innate immune function of lungs. Although alveolar type II cells cover only 5% of the alveolar surface area, these cells are metabolically active.¹⁴ They produce surfactants, serve as facultative progenitor cells to repair alveolar injury, and regulate innate immune function of the lung.¹⁴ These cells express Toll-like receptors (TLRs) and tumor necrosis factor receptors.¹⁵ Interactions with pathogens or endotoxins activate these receptors to initiate NF-κB signaling to produce tumor necrosis factor,¹⁶ IL-1 and IL-6,¹⁶ regulated on activation normal T cell expressed and secreted,¹⁷ and chemokine C-X-C motif ligand 1.¹⁸ These factors play key roles in recruiting inflammatory cells.^19–21 Alveolar type II cells also secrete the surfactant proteins (Sp)-A, -B, -C, and -D,²² which regulate innate and adaptive immunity by binding to antigen through interactions with surface receptors on inflammatory cell membranes.²³ Here, we studied the function of p120 through disrupting the p120 gene in alveolar type II cells in mice using the rtTA/TetO system coupled with a type II cell–specific SPC promoter. In these mice, we observed unchecked chronic lung inflammation associated with increased NF-κB activity and a persistently leaky alveolar epithelial barrier. These results provide the first genetic evidence that p120 in type II cells is a central regulator of innate immunity of lungs.     

Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.