Loss of Function of Intestinal IL-17 and IL-22 Producing Cells Contributes to Inflammation and Viral Persistence in SIV-Infected Rhesus Macaques

In HIV/SIV-infected humans and rhesus macaques (RMs), a severe depletion of intestinal CD4⁺ T-cells producing interleukin IL-17 and IL-22 associates with loss of mucosal integrity and chronic immune activation. However, little is known about the function of IL-17 and IL-22 producing cells during lentiviral infections. Here, we longitudinally determined the levels and functions of IL-17, IL-22 and IL-17/IL-22 producing CD4⁺ T-cells in blood, lymph node and colorectum of SIV-infected RMs, as well as how they recover during effective ART and are affected by ART interruption. Intestinal IL-17 and IL-22 producing CD4⁺ T-cells are polyfunctional in SIV-uninfected RMs, with the large majority of cells producing four or five cytokines. SIV infection induced a severe dysfunction of colorectal IL-17, IL-22 and IL-17/IL-22 producing CD4⁺ T-cells, the extent of which associated with the levels of immune activation (HLA-DR⁺CD38⁺), proliferation (Ki-67⁺) and CD4⁺ T-cell counts before and during ART. Additionally, Th17 cell function during ART negatively correlated with residual plasma viremia and levels of sCD163, a soluble marker of inflammation and disease progression. Furthermore, IL-17 and IL-22 producing cell frequency and function at various pre, on, and off-ART experimental points associated with and predicted total SIV-DNA content in the colorectum and blood. While ART restored Th22 cell function to levels similar to pre-infection, it did not fully restore Th17 cell function, and all cell types were rapidly and severely affected—both quantitatively and qualitatively—after ART interruption. In conclusion, intestinal IL-17 producing cell function is severely impaired by SIV infection, not fully normalized despite effective ART, and strongly associates with inflammation as well as SIV persistence off and on ART. As such, strategies able to preserve and/or regenerate the functions of these CD4⁺ T-cells central for mucosal immunity are critically needed in future HIV cure research.     

Expression of DC-SIGN by Dendritic Cells of Intestinal and Genital Mucosae in Humans and Rhesus Macaques

To better understand the role of dendritic cells (DCs) in human immunodeficiency virus (HIV) transmission at mucosal surfaces, we examined the expressions of the HIV adhesion molecule, dendritic-cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), its closely related homologue DC-SIGNR, and HIV coreceptors by distinct DC populations in the intestinal and genital tracts of humans and rhesus macaques. We also developed monoclonal antibodies (MAbs) specific for DC-SIGN or DC-SIGNR. In the Peyer's patches, DC-SIGN expression was detected in the interfollicular regions and in clusters of cells in the subepithelial dome regions. DC-SIGN expression was not found on plasmacytoid DCs. DC-SIGNR expression was restricted to endothelial cells in approximately one-third of the capillaries in the terminal ileum. In the vaginal epithelium, Langerhans' cells did not express DC-SIGN, whereas subepithelial DCs in the lamina propria expressed moderate levels of DC-SIGN. Finally, the rectum contained cells that expressed high levels of DC-SIGN throughout the entire thickness of the mucosa, while solitary lymphoid nodules within the rectum showed very little staining for DC-SIGN. Triple-color analysis of rectal tissue indicated that CCR5(+) CD4(+) DC-SIGN(+) DCs were localized just beneath the luminal epithelium. These findings suggest that DC-SIGN(+) DCs could play a role in the transmission of primate lentiviruses in the ileum and the rectum whereas accessibility to DC-SIGN(+) cells is limited in an intact vaginal mucosa. Finally, we identified a MAb that blocked simian immunodeficiency virus interactions with rhesus macaque DC-SIGN. This and other specific MAbs may be used to assess the relevance of DC-SIGN in virus transmission in vivo.     

Interleukin (IL)-17 enhances prostaglandin F(2 alpha)-stimulated IL-6 synthesis in osteoblasts

We previously showed that prostaglandin F(2alpha) (PGF(2alpha)) and endothelin-1 (ET-1) induce interleukin (IL)-6 through the activation of protein kinase C-dependent p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. It has recently been reported that tumor necrosis factor-alpha-induced IL-6 synthesis is amplified by IL-17 in these cells. In the present study, we investigated the effect of IL-17 on the IL-6 synthesis stimulated by PGF(2alpha) in MC3T3-E1 cells. IL-17 significantly enhanced the PGF(2alpha)-induced IL-6 synthesis in a dose-dependent manner in the range between 0.1 and 10 ng/ml. IL-17 also enhanced the IL-6 synthesis stimulated by 12- O -tetradecanoylphorbol-13-acetate, a direct activator of protein kinase C. In addition, IL-17 amplified the IL-6 synthesis induced by ET-1. However, IL-17 hardly affected the phosphorylation of p44/p42 MAP kinase induced by PGF(2alpha) or ET-1. These results strongly suggest that IL-17 enhances the IL-6 synthesis stimulated by PGF(2alpha) as well as ET-1 in osteoblasts, and that the effect is exerted at a point downstream from p44/p42 MAP kinase.     

Interleukin (IL)-12 mediates the anti-osteoclastogenic activity of CpG-oligodeoxynucleotides

Bacterial DNA activates the innate immune system via interactions with Toll-like receptor 9 (TLR9). This receptor recognizes CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the CpG dinucleotides in certain sequence contexts characterizing this DNA. Most studies have shown increased osteoclast differentiation by TLR ligands. We found that activation of TLRs (specifically TLR4 and TLR9) in early osteoclast precursors results in inhibition of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Our objective is to identify the mechanism leading to this inhibitory effect of a TLR ligand. Since both RANKL-RANK and CpG-ODN-TLR9 interactions result in NF-kappaB activation, p38 and ERK phosphorylation, and TNF-alpha synthesis (all implicated in osteoclastogenesis), we hypothesized that CpG-ODN (but not RANKL) in addition induces the synthesis of an anti-osteoclastogenic factor. Control osteoclast precursors, and cells treated with RANKL, CpG-ODN, or their combination were studied using DNA arrays (GEArray Q Series Mouse NF-kappaB Signaling Pathway Gene Array, MM-016, SuperArray). We found a marked increase in the mRNA levels of the osteoclastogenesis inhibitor interleukin-12 (IL-12) in osteoclast precursors treated with CpG-ODN and CpG-ODN + RANKL. Northern and Western analyses, together with ELISA, confirmed the DNA array studies. In correlation with these findings, IL-12 inhibited RANKL-induced osteoclast differentiation and specific anti-IL-12-antibodies inhibited the anti-osteoclastogenic effect of CpG-ODN. In conclusion, activation of TLR9 by its ligand, CpG-ODN, results in synthesis and release of IL-12 opposing RANKL-induced osteoclast differentiation.     

Alternative Activation of Macrophages in Rhesus Macaques (Macaca mulatta) with Endometriosis

Endometriosis is one of the most frequently encountered gynecologic diseases and a common cause of chronic pelvic pain and infertility. The pathophysiology of this syndrome can best be described as the presence of ectopic endometrium and a pelvic inflammatory process with associated immune dysfunction and alteration in the peritoneal environment. Macrophages play an important role in the progression and propagation of endometriosis. Alternative macrophage activation occurs in rodents and women with endometriosis but had not been examined previously in nonhuman primates. This case–control study aimed to characterize macrophage polarization in the ectopic and eutopic endometrial tissue of nonhuman primates with and without endometriosis. In addition, circulating cytokines in endometriosis cases and normal controls were investigated in an effort to identify serum factors that contribute to or result from macrophage polarization. Endometriosis lesions demonstrated increased infiltration by macrophages polarized toward the M2 phenotype when compared with healthy control endometrium. No serum cytokine trends consistent with alternative macrophage activation were identified. However, serum transforming growth factor α was elevated in macaques with endometriosis compared with healthy controls. Findings indicated that the activation state of macrophages in endometriosis tissue in nonhuman primates is weighted toward the M2 phenotype. This important finding enables rhesus macaques to serve as an animal model to investigate the contribution of macrophage polarization to the pathophysiology of endometriosis.Abbreviations: HLA, human leukocyte antigen; Iba1, ionized calcium binding adaptor molecule 1; M1, classically activated macrophage; M2, alternatively activated macrophage; sCD40L, soluble cluster of differentiation 40 ligand; TGF, transforming growth factor; VEGF, vascular endothelial growth factorEndometriosis is a common cause of chronic pelvic pain and infertility and affects more than 5.5 million women in North America alone.⁴¹ Although endometriosis is one of the most frequently encountered gynecologic health problems among women of reproductive age, the pathophysiology of this disease remains elusive due to its complexity and multifactorial etiology. The presence of functional endometrial glands and stroma outside the uterine cavity defines endometriosis. Currently, the most widely accepted theory for the origin of ectopic endometrial tissue is a combined effect of retrograde menstruation and associated implantation of endometrial fragments at an ectopic site. Progression of endometriosis lesions is thought to then be supported by peritoneal factors that allow cell adhesion and growth.⁴⁴ Although endometriosis is not a neoplastic disease, it exhibits aggressive features such as cellular proliferation, invasion, and vascular proliferation.¹² Strong evidence indicates that endometriosis involves a pelvic inflammatory process, with immune dysfunction and alteration in the peritoneal environment.^13,27 Numerous studies have demonstrated marked increases in macrophage populations and activity in the peritoneum of endometriosis patients.^6,54,59 Although macrophages are integral to homeostasis of the peritoneal environment, during endometriosis they mediate inflammation and facilitate the establishment and maintenance of the disease.Macrophages can be classified into 2 main populations: classically activated macrophages (M1), whose activating stimuli include IFNγ and LPS, and alternatively activated macrophages (M2), whose activating stimuli includes IL4, IL13, IL10, and transforming growth factor (TGF) β.⁵⁵ These polar phenotypes are not expressed together, but the activation state of tissue macrophages can change over time. This phenotypic switch is possible because macrophages retain plasticity, resulting in macrophage polarization that is transient and reversible.⁴⁰ A key component in determining the phenotype of the differentially activated macrophage is their response to microenvironmental signals, and this response allows for expression of a spectrum ranging from the M1 to M2 extremes.⁵¹ M1- and M2-activated macrophages perform different functions by producing pro- or antiinflammatory factors. M1 macrophages have enhanced endocytic functions and an enhanced ability to kill intracellular pathogens; they also secrete large amounts of proinflammatory cytokines such as IL1α, IL6, IL12, and TNFα.⁷ In contrast, M2 macrophages are involved in resolution of inflammation and promotion of tissue repair, and they secrete antiinflammatory and immunosuppressive cytokines including IL10 and TGFβ.³² M2 cells also express proangiogenic factors, such as coagulation factor XIII and vascular endothelial growth factor (VEGF) and have been associated with a high degree of vascularization in vivo.¹ The pathogenesis of endometriosis is therefore a likely combination of inappropriate or sustained polarization, leading to tissue damage (increased M1 response) and immune dysfunction (increased M2 response) and allowing for persistence of ectopic endometrial tissue.The use of animal models in endometriosis research is crucial. Work done with rodents involves the study of induced disease.⁵³ Despite this caveat, rodent models have been the basis for important contributions. Global macrophage depletion in a rat model of endometriosis effectively inhibits the initiation and growth of endometriosis implants.¹⁵ Attenuation of endometriosis has recently also been demonstrated in a mouse model of endometriosis.⁴ In that study, systemic depletion of macrophages was associated with failure of endometrial lesion development and defective angiogenesis of established lesions. Further evaluation of specific roles of differentially activated macrophages in that study⁴ showed that adoptive transfer of alternatively activated macrophages (M2) was associated with enhanced endometriosis progression. Conversely, adoptive transfer of inflammatory macrophages (M1) was associated with abrogated progression. In addition to evaluating murine lesions, the authors of the cited study⁴ investigated markers for alternative macrophage activation in women with endometriosis and matched controls which revealed increased expression of CD163 and CD206 (2 markers of M2 polarized macrophages) in endometriosis lesions as compared with disease-free peritoneum. Although many studies have been published about the pivotal role of macrophages in the pathophysiology of endometriosis, only a few have dealt with activation of the M1 and M2 macrophage phenotypes.^4,57 Furthermore, few studies have examined tissue infiltration of macrophages in eutopic endometrium of human subjects with endometriosis.^6,23 An exhaustive literature search failed to identify studies that investigate the role of M1 and M2 macrophage populations in eutopic endometrium.The current study uses rhesus macaques, which have been studied extensively in reproductive medicine.⁵⁸ Because spontaneous development of the disease requires menstrual shedding, endometriosis occurs naturally only in some nonhuman primate species, making development of lesions more comparable to the establishment of disease in humans.¹⁴ Compared with rodents, the nonhuman primate model of endometriosis is advantageous due to a close recapitulation of human disease and physiology. Work characterizing M1 and M2 macrophage activation in a species with spontaneous disease development may reflect a closer immunologic characterization to humans. In the current study, macrophage populations were evaluated in archival tissue collected from rhesus macaques with a diagnosis of endometriosis as confirmed by histologic examination. To characterize the phenotype of endometrial tissue macrophages in ectopic endometriosis lesions and eutopic endometrium of both cases and controls, immunohistochemistry was used to quantify cells expressing M1- and M2-specific markers. We hypothesized that endometriosis lesions and eutopic endometrium of rhesus macaques would be associated with a polarized macrophage infiltration consisting of increased numbers of M2 macrophages. This increase in M2 response may cause reduced immune clearance of ectopic endometrial cells, facilitating their implantation and growth. Further we speculated that M2 polarization would be associated with increased serum cytokines including IL10 and VEGF and decreased production of IL6, IL12, and TNFα. The lack of findings that support our hypotheses may suggest that the micro- or peritoneal environment is more important for lesion development or that another component of the systemic milieu is the determining factor in the development of endometriosis.     

Serum Interleukin (IL)-15 as a Biomarker of Alzheimer's Disease

Interleukin (IL-15), a pro-inflammatory cytokine has been studied as a possible marker of Alzheimer’s disease (AD); however its exact role in neuro-inflammation or the pathogenesis AD is not well understood yet. A Multiple Indicators Multiple Causes (MIMIC) approach was used to examine the relationship between serum IL-15 levels and AD in a well characterized AD cohort, the Texas Alzheimer''s Research and Care Consortium (TARCC). Instead of categorical diagnoses, we used two latent construct d (for dementia) and g’ (for cognitive impairments not contributing to functional impairments) in our analysis. The results showed that the serum IL-15 level has significant effects on cognition, exclusively mediated by latent construct d and g’. Contrasting directions of association lead us to speculate that IL-15’s effects in AD are mediated through functional networks as d scores have been previously found to be specifically related to default mode network (DMN). Our finding warrants the need for further research to determine the changes in structural and functional networks corresponding to serum based biomarkers levels.     

Interleukin (IL)-17 enhances tumor necrosis factor-alpha-stimulated IL-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

Inflammatory cytokines are well known to play crucial roles in the pathogenesis of rheumatoid arthritis. Among them, interleukin (IL)-17 is a cytokine that is mainly synthesized by activated T cells and its receptors are present in osteoblasts. The synthesis of IL-6, known to stimulate osteoclastic bone resorption, is reportedly responded to bone resorptive agents such as tumor necrosis factor-alpha (TNF-alpha) in osteoblasts. It has been reported that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We previously showed that sphingosine 1-phosphate (S1-P) mediates TNF-alpha-stimulated IL-6 synthesis in these cells. In the present study, we investigated the mechanism of IL-17 underlying enhancement of IL-6 synthesis in MC3T3-E1 cells. IL-17 induced phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the enhancement by IL-17 of TNF-alpha-stimulated IL-6 synthesis. IL-17 also amplified S1-P-stimulated IL-6 synthesis, and the amplification by IL-17 was suppressed by SB203580. Anisomycin, an activator of p38 MAP kinase, which alone had no effect on IL-6 level, enhanced the IL-6 synthesis stimulated by TNF-alpha. SB203580 and PD169316 inhibited the amplification by anisomycin of the TNF-alpha-induced IL-6 synthesis. Taken together, our results strongly suggest that IL-17 enhances TNF-alpha-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.     

Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4⁺ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.     

Endometrial Decidualization and Deciduosis in Aged Rhesus Macaques (Macaca mulatta)

Superficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates. Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells. Previous reports involving nonpregnant rhesus monkeys have described localized and widespread endometrial decidualization in response to administration of progesterone and synthetic progestogens. Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells are located outside of the endometrium, most often in the ovaries, uterus and cervix but also in various other organs. In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua can be found in the ovary in nearly all term pregnancies. Here we describe pronounced endometrial decidualization in 2 rhesus macaques. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. In one animal, florid extrauterine and peritoneal serosal decidualization was admixed multifocally with carcinomatosis from a primary colonic adenocarcinoma. Cells constituting endometrial and serosal decidualization reactions were immunopositive for the stromal markers CD10, collagen IV, smooth muscle actin, and vimentin and immunonegative for cytokeratin. In contrast, carcinomatous foci were cytokeratin-positive. To our knowledge, this report describes the first cases of serosal peritoneal decidualization in rhesus macaques. The concurrent presentation of serosal peritoneal decidualization with carcinomatosis is unique.Abbreviations: GnRH, gonadotropin-releasing hormone; PAS, periodic acid–Schiff; SMA, smooth-muscle actinSuperficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates.^13,27,29,37 This process typically begins, and is most prominent, adjacent to the spiral arteries, eventually expanding to affect the endometrium uniformly.³⁵ The endometrial stroma surrounds and supports the endometrial glands and is composed mainly of endometrial stromal cells and blood vessels.³⁵ Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells.^7,27,38 Endometrial stromal cells transform into large, polyhedral, cytoplasm-rich cells with large amounts of stored glycogen and are often binucleated or polyploid in character.^{6,13,27,30,35} Ultrastructurally, decidualized cells have numerous ribosomes, prominent rough endoplasmic reticulum and Golgi complexes, and cytoplasmic accumulation of glycogen and lipid droplets.^13,35 Consistent with their stromal origin, decidualized cells express mesenchymal immunohistochemical markers, such as vimentin, desmin, and muscle-specific actin.^{6,7,14,16,20,22}Initiation of decidualization by attachment of the blastocyst to the uterine epithelium depends on previous sensitization by progesterone secretion, after a brief priming by estrogen.^12,13,27 Estrogen and progesterone regulate a series of complex interactions at the interface between the developing embryo and the cells in the stromal compartment, leading to the formation of a differentiated maternal tissue (decidua) that supports embryo growth and maintains early pregnancy.²⁷ Postovulatory levels of circulating progesterone increase and help maintain the differentiation of decidual cells.^{7,13,33,37,38}Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells reside outside of the endometrium, most often in the ovaries, uterus, and cervix; the fallopian tubes, peritoneum, omentum, diaphragm, liver, skin, spleen, appendix, abdominal–pelvic lymph nodes, renal pelvis, and lungs of women have also been reported as affected.^{6,14,18,20,22,28,29,38} In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua have been reported in the ovary in 90.5% to 100% of term pregnancies.^{6-8,14,20,22,28-30,38} Occasional cases in nonpregnant or postmenopausal women have been attributed to progesterone-secreting active corpora lutea, progesterone secretion by the adrenal cortex, trophoblastic disease, exogenous progestational agents, and pelvic irradiation.^{6-8,14,18,20,22,28,38} Deciduosis is usually an incidental finding that regresses postpartum within 4 to 6 wk; rarely, florid reactions have been reported to cause peritonitis, adhesions, hydronephrosis and hematuria, acute bowel obstruction or perforation (or both), abdominal pain mimicking appendicitis, massive and occasionally fatal hemoperitoneum, vaginal bleeding, and pneumothorax.^{6,7,14,18,20,22,28,29,31}Previous reports involving nonpregnant rhesus macaques have described localized and widespread endometrial decidualization in response to the administration of progesterone, synthetic progestogens, or progesterone-releasing bioactive intrauterine devices and intravaginal rings and have referred to these changes as ‘pseudodecidualization’ to indicate the absence of pregnancy in these animals.^12,33,35,37 In macaques given low (but superphysiologic) levels of progestogens, decidual changes have been noted in localized regions (around spiral arteries and underneath superficial epithelium), whereas high doses of progesterone or synthetic progestagens can cause a more pronounced and extensive reaction.³⁵In cynomolgus macaques, extrauterine decidual cell plaques are rare histologic findings in the subcoelomic mesenchyme of the ovarian cortex.^8,30 Despite the frequency of the condition in women, deciduosis is postulated to be a rarely documented lesion in primates because it is most often observed in conjunction with pregnancy, and pregnant cynomolgus macaques are seldom used in toxicity studies.⁸ Here we describe the pronounced endometrial decidualization of 2 rhesus macaques, one of which also had florid extrauterine and peritoneal decidualization that was admixed multifocally with carcinomatosis. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. To our knowledge, this report describes the first cases of peritoneal decidualization in rhesus macaques as well as the concurrent occurrence of carcinomatosis, endometriosis and peritoneal decidualization in a macaque. The extensive intermixing of the cell populations presented a diagnostic challenge at pathologic examination, and accurate diagnosis was achieved only through the use of multiple immunohistochemical markers.     

Efficacy of Oral Fecal Bacteriotherapy in Rhesus Macaques (Macaca mulatta) with Chronic Diarrhea

Chronic diarrhea remains the principal burden in providing health care for nonhuman primates in biomedical research facilities. Although the exact etiology continues to puzzle nonhuman primate clinicians, recent research in humans has shown that restoring the indigenous microbial diversity may be successful in resolving cases of chronic diarrhea when other treatment modalities have failed. The process of restoring this microbial balance, known as fecal bacteriotherapy, uses the complete flora from a normal donor as a therapeutic probiotic mixture. In the current study, Indian-origin rhesus macaques were randomized into treatment (n = 7) and control (n = 6) groups to determine whether orally administered fecal bacteriotherapy would reduce the overall incidence of chronic diarrhea during a 60-d follow-up period in the treatment group compared with control macaques, which received a placebo. Although the treatment effect, determined by comparing the baseline fecal scores of the treatment and control groups, did not reach statistical significance, preprocedure and postprocedure fecal scores in the treatment group differed significantly. These findings are encouraging, and we hope that our study will motivate larger studies evaluating the use of fecal bacteriotherapy in nonhuman primates.Chronic diarrhea is perhaps the most daunting clinical challenge of nearly every biomedical research facility that houses large numbers of nonhuman primates. Our facility, The Oregon National Primate Research Center (ONPRC), is no exception. As of 2008, approximately 14.7% of the total population at this center was reported to have diarrhea requiring medical attention each year, which constituted an average of 35.2% of the total clinical caseload.²⁷ Our more recent analysis of the medical records from 2010 has confirmed these statistics: 16.2% of the total population was treated for diarrhea in 2010, which comprised 29.3% of the total clinical caseload for that year. The cost of chronic diarrhea to institutions such as ours in terms of veterinary staff time, diagnostics, and medications is profound. Consequently, colony management personnel and resources are taxed due to the care and maintenance of these patients, given that nonhuman primates with chronic diarrhea generally are in poor body condition, lag behind the growth rate of their peers, and require frequent medical intervention, thereby making them undesirable as research subjects and unproductive members of the breeding colony.⁸Chronic diarrhea is undeniably the largest, most expensive problem in providing health care for nonhuman primate colonies. Nonhuman primates diagnosed with chronic diarrhea typically test negative for known fecal pathogens^19,24,27 and are recalcitrant to common diarrhea treatment modalities. For these reasons, the underlying cause of chronic diarrhea has been elusive and is likely multifactorial. Over the years, numerous researchers and clinicians in this field have attempted to devise an effective treatment regimen for these patients, with little success.Recent research in humans has shown that restoring the indigenous microbial diversity may be useful in resolving cases of chronic diarrhea when other treatment modalities have failed.^10,18 A normal healthy digestive tract contains numerous bacterial inhabitants, which typically act to impede exogenous bacteria from establishing themselves as pathogens. After an episode of gastrointestinal disease that results in diarrhea, the population of indigenous bacteria often is disrupted, subsequently leading to decreased numbers and diversity of these organisms. This imbalance, or dysbiosis, may result from pathogenic diarrhea or may be nosocomial due to prescribed antibiotic therapy.^7,10,18,21 Several publications have explored the idea that dysbiosis can be treated with an infusion of normal flora.^1,4,16 Fecal bacteriotherapy uses the complete flora of a normal donor as a therapeutic probiotic mixture of living organisms.⁵ Because the bacterial components of the normal fecal flora that are the most important for host defense are unknown, reintroducing all flora is currently recommended.²³ In addition to providing the complete bacterial flora from a normal donor, another possible advantage of this therapy is that it halts the cycle of antimicrobial use in these patients.¹ The discontinuance of intestinal flora disruption through the use of antimicrobials, when combined with the probiotic effects of fecal bacteriotherapy, constitutes the philosophy of this therapeutic approach. Several case series in the human literature have demonstrated that this therapy is capable of resolving refractory cases of diarrhea, with very high success rates after single administrations.^2,4,11,15,28 In addition, the transplantation of donor stool can dramatically change the recipient''s intestinal flora in as little as 14 d.¹⁶ Furthermore, fecal bacteriotherapy has been an effective tool in veterinary medicine for the treatment of ruminants and horses with enteric disease.^6,9,12,22 However, whether this treatment modality will be effective in nonhuman primates or whether successful cases will continue to be sporadic and species-specific remains unknown.The goal of the current study was to test a new treatment modality, fecal bacteriotherapy, which if successful, would reduce the overall incidence of chronic diarrhea in rhesus macaques. Because the need for detailed information regarding techniques used to prepare and administer the fecal suspension has been recognized in the human literature,^5,23,28 we here describe in detail the standardized treatment protocol that we developed. Our hypothesis was that the incidence and severity of diarrhea during the 60-d follow-up period would be decreased in macaques that received fecal bacteriotherapy compared with those that received placebo treatment.     

Rhesus Macaques Infected with Macrophage-Tropic Simian Immunodeficiency Virus (SIVmacR71/17E) Exhibit Extensive Focal Segmental and Global Glomerulosclerosis

We previously showed that inoculation of rhesus macaques with molecularly cloned lymphocytetropic simian immunodeficiency virus (SIV_mac239) results in SIV-associated nephropathy (SIVAN) and that the glomerulosclerotic lesions were associated with the selection of macrophagetropic (M-tropic) variants (V. H. Gattone et al., AIDS Res. Hum. Retroviruses 14:1163–1180, 1998). In the present study, seven rhesus macaques were inoculated with M-tropic SIV_macR71/17E, and the renal pathology was examined at necropsy. All SIV_macR71/17E-infected macaques developed AIDS, and most developed other systemic complications, including SIV-induced encephalitis and lentivirus interstitial pneumonia. There was no correlation between the length of infection (42 to 97 days), circulating CD4⁺ T-cell counts, and renal disease. Of the seven macaques inoculated with SIV_macR71/17E, five developed significant mesangial hyperplasia and expansion of matrix and four were clearly azotemic (serum urea nitrogen concentration of 40 to 112 mg/dl). These same five macaques developed focal segmental to global glomerulosclerotic lesions. Increased numbers of glomerular CD68⁺ cells (monocytes/macrophages) were found in glomeruli but not the tubulointerstitium of the macaques inoculated with SIV_macR71/17E. All macaques had glomerular deposits of immunoglobulin G (IgG), IgM, and tubuloreticular inclusions, and six of seven had IgA deposition. However, there was no correlation between the presence of circulating anti-SIV_mac antibodies, immunoglobulin deposition, and glomerular disease. Tubulointerstitial infiltrates were mild, with little or no correlation to azotemia, while microcystic tubules were evident in those with glomerulosclerosis or azotemia. The four most severely affected macaques were positive for diffuse glomerular immunostaining for viral core p27 antigen, and there was intense staining in the glomeruli of the two macaques with the most severe glomerulosclerosis. Viral sequences were isolated from glomerular and tubulointerstitial fractions from macaques with severe glomerulosclerosis but only from the tubulointerstitial compartment of those that did not develop glomerulosclerosis. Interviral recombinant viruses generated with env sequences isolated from glomeruli confirmed the M-tropic nature of the virus found in the glomeruli. The correlation between the increased number of CD68⁺ cells (monocytes/macrophages) in the glomeruli, the localization of p27 antigen in the glomeruli, and the glomerular pathology confirms and extends our previous observations of an association between glomerular infection and infiltration by M-tropic virus and SIVAN.     

Implication of Interleukin (IL)-18 in the pathogenesis of chronic obstructive pulmonary disease (COPD)

Interleukin (IL)-18 is a pro-inflammatory cytokine that was firstly described as an interferon (IFN)-γ-inducing factor. Similar to IL-1β, IL-18 is synthesized as an inactive precursor requiring processing by caspase-1 into an active cytokine. The platform for activating caspase-1 is known as the inflammasome, a multiple protein complex. Macrophages and dendritic cells are the primary sources for the release of active IL-18, whereas the inactive precursor remains in the intracellular compartment of mesenchymal cells. Finally, the IL-18 precursor is released from dying cells and processed extracellularly.IL-18 has crucial host defense and antitumor activities, and gene therapy to increase IL-18 levels in tissues protects experimental animals from infection and tumor growth and metastasis. Moreover, multiple studies in experimental animal models have shown that IL-18 over-expression results to emphysematous lesions in mice. The published data prompt to the hypothesis that IL-18 induces a broad spectrum of COPD-like inflammatory and remodeling responses in the murine lung and also induces a mixed type 1, type 2, and type 17 cytokine responses. The majority of studies identify IL-18 as a potential target for future COPD therapeutics to limit both the destructive and remodeling processes occurring in COPD lungs.     

Examining the Species-Specificity of Rhesus Macaque Cytomegalovirus (RhCMV) in Cynomolgus Macaques

Cytomegalovirus (CMV) is a highly species-specific virus that has co-evolved with its host over millions of years and thus restricting cross-species infection. To examine the extent to which host restriction may prevent cross-species research between closely related non-human primates, we evaluated experimental infection of cynomolgus macaques with a recombinant rhesus macaque-derived CMV (RhCMV-eGFP). Twelve cynomolgus macaques were randomly allocated to three groups: one experimental group (RhCMV-eGFP) and two control groups (UV-inactivated RhCMV-eGFP or media alone). The animals were given two subcutaneous inoculations at week 0 and week 8, and a subset of animals received an intravenous inoculation at week 23. No overt clinical or haematological changes were observed and PBMCs isolated from RhCMV-eGFP inoculated animals had comparable eGFP- and IE-1-specific cellular responses to the control animals. Following inoculation with RhCMV-eGFP, we were unable to detect evidence of infection in any blood or tissue samples up to 4 years post-inoculation, using sensitive viral co-culture, qPCR, and Western blot assays. Co-culture of urine and saliva samples demonstrated the presence of endogenous cynomolgus CMV (CyCMV) cytopathic effect, however no concomitant eGFP expression was observed. The absence of detectable RhCMV-eGFP suggests that the CyCMV-seropositive cynomolgus macaques were not productively infected with RhCMV-eGFP under these inoculation conditions. In a continued effort to develop CMV as a viral vector for an HIV/SIV vaccine, these studies demonstrate that CMV is highly restricted to its host species and can be highly affected by laboratory cell culture. Consideration of the differences between lab-adapted and primary viruses with respect to species range and cell tropism should be a priority in evaluating CMV as vaccine vector for HIV or other pathogens at the preclinical development stage.     

Correlates of Male Consortship Rate in Free-Ranging Rhesus Macaques (Macaca mulatta)

Male–male competition for access to receptive females can take the form of nonrecurring fights and/or a sustained contest over mating opportunities. Male physical condition has been linked to dominance rank and reproductive success in species characterized by intrasexual fights for dominance and access to females. In group-living species characterized by endurance rivalry, however, factors contributing to male reproductive success are less well understood. In such species, particularly seasonally breeding ones with low sexual dimorphism and seniority-based rank, age and social factors other than rank may prove important. In the absence of genetic data, male mate guarding or consortship can serve as an indicator of male reproductive success. To evaluate the contribution of age and intragroup sociality to male consortship rate, I collected behavioral data during one nonmating and one mating season in two social groups of free-ranging rhesus macaques that experience no predation or food scarcity. Higher-ranking males, younger males, or males that exhibited lower rate of intrasexual aggression had higher consortship rates. Male–female dyads that groomed outside consortship did not form consortships as often as dyads that did not engage in nonconsort grooming. This is the first study to identify the significance of male–male aggression and male–female affiliation to male consortship rate in a species characterized by endurance rivalry, high male rank stability, and strong female mate choice. Social behaviors and male age may be particularly important in determining male reproductive success in populations experiencing high food availability and a lack of predation, which are typical of an increasing number of vertebrates in areas densely populated by humans.     

Hematology and Culture Assessment of Cranially Implanted Rhesus Macaques (Macaca mulatta)

The use of percutaneous cranial implants in rhesus macaques (Macaca mulatta) has long been a valuable tool for neuroscience research. However, when treating and assessing these animals, veterinarians are required to make assumptions about diagnostic results due to a lack of research into how these implants affect physiology. Microbial cultures of cranial implant sites show an abundance of colonizing bacteria, but whether these microbes affect animal health and wellbeing is poorly understood. In addition, microbial antibiotic resistance can present significant health concerns for both the animals and the researchers. To help elucidate the relationship between percutaneous cranial implants and blood parameters, complete blood cell counts and serum chemistry results were assessed on 57 nonhuman primates at our institution from September 2001 to March 2017. Generalized estimating equations were used to compare the results before and after an animal''s first implant surgery. This modelling showed that cranial implants were a significant predictor of alterations in the number of neutrophils, lymphocytes, and red blood cells, and in the concentration of hemoglobin, alkaline phosphatase, creatinine, calcium, phosphorus, total protein, albumin, and globulin. Anaerobic and aerobic bacterial cultures were performed to identify bacteria associated with cranial implants. Staphylococcus spp., Streptococcus spp., and Corynebacterium spp. comprised the majority of the aerobic bacterial isolates, while Fusobacterium spp., Peptostreptococcus spp. and Bacterioides fragilis comprised the majority of anaerobic bacterial isolates. Using a Pearson r correlation for statistical analysis, we assessed whether any of these bacterial isolates developed antibiotic resistances over time. Cefazolin, the most frequently used antibiotic in monkeys in this study, was the only antimicrobial out of 41 agents tested to which bacteria developed resistance over time. These results indicate that percutaneous implants are associated with a generalized inflammatory state, multiple bacterial species are present at the implant site, and these bacteria may contribute to the inflammatory response.

Neuroscience research often employs in vivo analysis of neuronal distribution, activity, and function, all of which require visualization and manipulation of the brain in living animals. To achieve this, cortical structures are accessed by the placement of percutaneous implants that are anchored to the skull and outfitted with transcranial ports. Studies of the auditory system,^72,78 visual cortex,^20,22,46,54 motor cortex,^23,44 perception,⁷⁰ and optogenetics^23,80 have all benefitted from the use of these implants in multiple laboratory species. However, few studies have investigated the effects of these implants on the general physiology of research animals.³²Due to human similarities in neuroanatomy, physiology, social behavior, and cognition, rhesus macaques (Macaca mulatta) have proven invaluable in translational studies of cortical structures. This species has long been the recipient of percutaneous cranial implants²⁹ and therefore, has been the subject of many refinements in their construction to improve research outcomes.^{3,11,40,43,60} Traditionally, these implants are constructed out of titanium or titanium alloy hardware that is anchored in place with acrylic or bone cement. Recording chambers provide access points through craniotomy sites where devices are implanted into deeper structures of the brain. Titanium alloys have been shown to provide a good bone/implant interface through the development of titanium oxides;^17,32,59 however, these implants may leach ions into surrounding tissues, with unknown health or research implications.³² Acrylics provide a moldable and easily repairable substrate, but have poor biocompatibility.^5,53 Their addition to implants increases exposed tissue surface area, creating ideal environments for infectious agents to thrive.^2,43Cranial implants can develop complications, including inflammation and infections at skin margins and the bone/implant interface.³² Multiple species of bacteria, including Staphylococcus spp., Corynebacterium spp., Enterococcus spp., and Providencia rettgeri, have been recovered from the chambers and skin edges of cranial implants in rhesus macaques.^10,43,88 These infections may be due to manipulation and contamination of implants by the animals themselves. Chronicity of infections is also likely due to the inability to apply therapeutic agents to deeper regions of tissue shielded by the implant. Chronic pathology is evident in deeper structures at the neural-implant interface.⁶⁷ For example, sustained glial responses and neural degeneration have been identified at sites where electrodes and arrays contact brain tissue.^12,74Chronic inflammation has well-established effects on hematologic values, such as red and white blood cell counts, platelet numbers, and total protein measurements. Few studies are currently available that link chronic percutaneous implants to changes in hematology in rhesus macaques, requiring clinicians to use established reference intervals of nonimplanted animals when analyzing bloodwork. This requires assumptions about effects of implants on overall health; these assumptions may or may not be correct, but are the necessary basis for assessments and therapies. In this study, systemic physiologic changes were identified in clinically healthy, implanted animals to provide a framework for analysis of complete blood cell counts (CBC) and serum chemistry (CHEM) values for clinicians working with rhesus macaques with percutaneous implants. We also assess bacterial cultures taken from these implants to identify common contaminants and their antibiotic resistance profiles. We hypothesize that clinically healthy rhesus macaques with percutaneous implants have significantly different hematology profiles than those without implants, despite a lack of clinical signs. By understanding how implants and associated bacteria affect routine blood test results, observed changes can be better characterized as normal or abnormal, so that veterinary care can be tailored as needed. This important step will allow improved care and welfare of animals used in neuroscience research.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.