Matthew J. Kan

Arginine Deprivation and Immune Suppression in a Mouse Model of Alzheimer's Disease

The pathogenesis of Alzheimers disease (AD) is a critical unsolved question; and although recent studies have demonstrated a strong association between altered brain immune responses and disease progression, the mechanistic cause of neuronal dysfunction and death is unknown. We have previously described the unique CVN-AD mouse model of AD, in which immune-mediated nitric oxide is lowered to mimic human levels, resulting in a mouse model that demonstrates the cardinal features of AD, including amyloid deposition, hyperphosphorylated and aggregated tau, behavioral changes, and age-dependent hippocampal neuronal loss. Using this mouse model, we studied longitudinal changes in brain immunity in relation to neuronal loss and, contrary to the predominant view that AD pathology is driven by proinflammatory factors, we find that the pathology in CVN-AD mice is driven by local immune suppression. Areas of hippocampal neuronal death are associated with the presence of immunosuppressive CD11c+ microglia and extracellular arginase, resulting in arginine catabolism and reduced levels of total brain arginine. Pharmacologic disruption of the arginine utilization pathway by an inhibitor of arginase and ornithine decarboxylase protected the mice from AD-like pathology and significantly decreased CD11c expression. Our findings strongly implicate local immune-mediated amino acid catabolism as a novel and potentially critical mechanism mediating the age-dependent and regional loss of neurons in humans with AD.

Macrophages Contribute to the Spermatogonial Niche in the Adult Testis

The testis produces sperm throughout the male reproductive lifespan by balancing self-renewal and differentiation of spermatogonial stem cells (SSCs). Part of the SSC niche is thought to lie outside the seminiferous tubules of the testis; however, specific interstitial components of the niche that regulate spermatogonial divisions and differentiation remain undefined. We identified distinct populations of testicular macrophages, one of which lies on the surface of seminiferous tubules, in close apposition to areas of tubules enriched for undifferentiated spermatogonia. These macrophages express spermatogonial proliferation- and differentiation-inducing factors, such as colony-stimulating factor 1 (CSF1) and enzymes involved in retinoic acid (RA) biosynthesis. We show that transient depletion of macrophages leads to a disruption in spermatogonial differentiation. These findings reveal an unexpected role for macrophages in the spermatogonial niche in the testis and raise the possibility that macrophages play previously unappreciated roles in stem/progenitor cell regulation in other tissues.

Identification of a Tissue-Specific, C/EBPβ-Dependent Pathway of Differentiation for Murine Peritoneal Macrophages

Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are largely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-β. Cebpb−/− mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb−/− mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb−/− mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBPβ-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBPβ plays an intrinsic, tissue-restricted role in the generation of resident macrophages.

iNKT Cells Require CCR4 to Localize to the Airways and to Induce Airway Hyperreactivity

iNKT cells are required for the induction of airway hyperreactivity (AHR), a cardinal feature of asthma, but how iNKT cells traffic to the lungs to induce AHR has not been previously studied. Using several models of asthma, we demonstrated that iNKT cells required the chemokine receptor CCR4 for pulmonary localization and for the induction of AHR. In both allergen-induced and glycolipid-induced models of AHR, wild-type but not CCR4−/− mice developed AHR. Furthermore, adoptive transfer of wild-type but not CCR4−/− iNKT cells reconstituted AHR in iNKT cell-deficient mice. Moreover, we specifically tracked CCR4−/− vs wild-type iNKT cells in CCR4−/−:wild-type mixed BM chimeric mice in the resting state, and when AHR was induced by protein allergen or glycolipid. Using this unique model, we showed that both iNKT cells and conventional T cells required CCR4 for competitive localization into the bronchoalveolar lavage/airways compartment. These results establish for the first time that the pulmonary localization of iNKT cells critical for the induction of AHR requires CCR4 expression by iNKT cells.

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

Natural genetic variation of integrin alpha L (Itgal) modulates ischemic brain injury in stroke.

During ischemic stroke, occlusion of the cerebrovasculature causes neuronal cell death (infarction), but naturally occurring genetic factors modulating infarction have been difficult to identify in human populations. In a surgically induced mouse model of ischemic stroke, we have previously mapped Civq1 to distal chromosome 7 as a quantitative trait locus determining infarct volume. In this study, genome-wide association mapping using 32 inbred mouse strains and an additional linkage scan for infarct volume confirmed that the size of the infarct is determined by ancestral alleles of the causative gene(s). The genetically isolated Civq1 locus in reciprocal recombinant congenic mice refined the critical interval and demonstrated that infarct size is determined by both vascular (collateral vessel anatomy) and non-vascular (neuroprotection) effects. Through the use of interval-specific SNP haplotype analysis, we further refined the Civq1 locus and identified integrin alpha L (Itgal) as one of the causative genes for Civq1. Itgal is the only gene that exhibits both strain-specific amino acid substitutions and expression differences. Coding SNPs, a 5-bp insertion in exon 30b, and increased mRNA and protein expression of a splice variant of the gene (Itgal-003, ENSMUST00000120857), all segregate with infarct volume. Mice lacking Itgal show increased neuronal cell death in both ex vivo brain slice and in vivo focal cerebral ischemia. Our data demonstrate that sequence variation in Itgal modulates ischemic brain injury, and that infarct volume is determined by both vascular and non-vascular mechanisms.

C-C Motif Chemokine 5 Attenuates Angiotensin II-Dependent Kidney Injury by Limiting Renal Macrophage Infiltration.

Inappropriate activation of the renin angiotensin system (RAS) is a key contributor to the pathogenesis of essential hypertension. During RAS activation, infiltration of immune cells into the kidney exacerbates hypertension and renal injury. However, the mechanisms underpinning the accumulation of mononuclear cells in the kidney after RAS stimulation remain unclear. C-C motif chemokine 5 (CCL5) drives recruitment of macrophages and T lymphocytes into injured tissues, and we have found that RAS activation induces CCL5 expression in the kidney during the pathogenesis of hypertension and renal fibrosis. We therefore evaluated the contribution of CCL5 to renal damage and fibrosis in hypertensive and normotensive models of RAS stimulation. Surprisingly, during angiotensin II-induced hypertension, CCL5-deficient (knockout, KO) mice exhibited markedly augmented kidney damage, macrophage infiltration, and expression of proinflammatory macrophage cytokines compared with wild-type controls. When subjected to the normotensive unilateral ureteral obstruction model of endogenous RAS activation, CCL5 KO mice similarly developed more severe renal fibrosis and greater accumulation of macrophages in the kidney, congruent with enhanced renal expression of the macrophage chemokine CCL2. In turn, pharmacologic inhibition of CCL2 abrogated the differences between CCL5 KO and wild-type mice in kidney fibrosis and macrophage infiltration after unilateral ureteral obstruction. These data indicate that CCL5 paradoxically limits macrophage accumulation in the injured kidney during RAS activation by constraining the proinflammatory actions of CCL2.

Whole-genome sequencing of Atacama skeleton shows novel mutations linked with dysplasia

Over a decade ago, the Atacama humanoid skeleton (Ata) was discovered in the Atacama region of Chile. The Ata specimen carried a strange phenotype-6-in stature, fewer than expected ribs, elongated cranium, and accelerated bone age-leading to speculation that this was a preserved nonhuman primate, human fetus harboring genetic mutations, or even an extraterrestrial. We previously reported that it was human by DNA analysis with an estimated bone age of about 6-8 yr at the time of demise. To determine the possible genetic drivers of the observed morphology, DNA from the specimen was subjected to whole-genome sequencing using the Illumina HiSeq platform with an average 11.5× coverage of 101-bp, paired-end reads. In total, 3,356,569 single nucleotide variations (SNVs) were found as compared to the human reference genome, 518,365 insertions and deletions (indels), and 1047 structural variations (SVs) were detected. Here, we present the detailed whole-genome analysis showing that Ata is a female of human origin, likely of Chilean descent, and its genome harbors mutations in genes (COL1A1, COL2A1, KMT2D, FLNB, ATR, TRIP11, PCNT) previously linked with diseases of small stature, rib anomalies, cranial malformations, premature joint fusion, and osteochondrodysplasia (also known as skeletal dysplasia). Together, these findings provide a molecular characterization of Atas peculiar phenotype, which likely results from multiple known and novel putative gene mutations affecting bone development and ossification.

Nanoparticle formulation improves doxorubicin efficacy by enhancing host antitumor immunity

Abstract Strategies that enhance the host antitumor immune response promise to revolutionize cancer therapy. Optimally mobilizing the immune system will likely require a multi‐pronged approach to overcome the resistance developed by tumors to therapy. Recently, it has become recognized that doxorubicin can contribute to re‐establishing host antitumor immunity through the generation of immunogenic cell death. However, the potential for delivery strategies to further enhance the immunological effects of doxorubicin has not been adequately examined. We report herein that Chimeric Polypeptide Doxorubicin (CP‐Dox), a nanoparticle formulation of doxorubicin, enhances antitumor immunity. Compared to free doxorubicin, a single intravenous (IV) administration of CP‐Dox at the maximum tolerated dose increases the infiltration of leukocytes into the tumor, slowing tumor growth and preventing metastasis in poorly immunogenic 4T1 mammary carcinoma. We demonstrate that the full efficacy of CP‐Dox is dependent on CD8 + T cells and IFN‐&ggr;. CP‐dox treatment also repolarized intratumoral myeloid cells towards an antitumor phenotype. These findings demonstrate that a nanoparticle drug is distinct from the free drug in its ability to productively stimulate antitumor immunity. Our study strongly argues for the use of antitumor immunotherapies combined with nanoparticle‐packaged chemotherapy. Graphical abstract Model of CP‐Doxs enhancement of the host antitumor immune response in 4T1 mammary carcinoma. CP‐Dox treatment generates inflammatory chemokines and IFN‐&ggr;. Chemokine production leads to the recruitment of leukocytes including CD8 + T cells and inflammatory monocytes. CD8 + T cells, in the presence of increased IFN‐&ggr;, contribute to controlling the primary tumor and preventing metastasis. Figure. No Caption available.