Patcharin Pramoonjago

Hepatocellular ballooning in NASH

BACKGROUND & AIMS Hepatocellular ballooning is a key finding in nonalcoholic steatohepatitis (NASH). It is conventionally defined by hemotoxylin and eosin (H&E) staining showing enlarged cells with rarefied cytoplasm and recently by changes in the cytoskeleton. Fat droplets are emerging as important organelles in cell metabolism. To address a possible relation between fat droplets and ballooning, we studied fat staining, H&E, and keratin 18 staining in human NASH. METHODS Sequential staining and high resolution imaging were used to study freshly prepared cryo-sections from 10 patients with histologically confirmed steatohepatitis using oil red O for fat droplet identification, H&E to identify ballooning, and anti-K18 to confirm cytoskeletal changes. High resolution images were captured at each stage using the Aperio Scanscope. To provide ultrastructural correlation, glutaraldehyde-fixed specimens were studied using transmission electron microscopy (TEM) with serial sectioning for localization of ballooned cells by light microscopy and TEM in identical specimens. RESULTS Serial staining consistently demonstrated that hepatocellular ballooning is associated with fat droplet accumulation evident by oil red O positivity and depletion of cytoplasmic keratin 18 with K-18 positive Mallory-Denk bodies (MDB). TEM confirmed the association between osmium stained fat droplets, MDB formation, and cellular enlargement and suggested droplet-associated dilation of the endoplasmic reticulum. CONCLUSIONS These results indicate a relationship between cellular ballooning, fat droplet accumulation, and cytoskeletal injury in NASH. We speculate that injury to multiple, organelles including fat droplets and endoplasmic reticulum, contribute to this characteristic finding.

The role of physiological self-antigen in the acquisition and maintenance of regulatory T-cell function

Summary:  The CD4+CD25+ regulatory T cells (Tregs) are efficient regulators of autoimmunity, but the mechanism remains elusive. We summarize recent data for the conclusion that disease‐specific Tregs respond to tissue antigens to maintain physiological tolerance and prevent autoimmunity. First, polyclonal Tregs from antigen‐positive donors suppress autoimmune ovarian disease (AOD) or experimental autoimmune prostatitis in day 3 thymectomized (d3tx) mice more efficiently than Tregs from antigen‐negative donors. Second, Tregs of antigen‐negative adult mice respond to cognate antigen in vivo and rapidly gain disease‐specific Treg function. Third, in d3tx female recipients devoid of neonatal ovarian antigens, only female Tregs suppressed AOD; the male Tregs gain AOD‐suppressing function by responding to the ovarian antigen in the recipients and mask the supremacy of female Tregs in AOD suppression. Fourth, when Tregs completely suppress AOD, the ovary‐draining lymph node is the only location with evidence of profound and persistent (but reversible) host T‐cell suppression. Fifth, from these nodes, highly potent AOD‐suppressing Tregs are retrievable. We conclude that self‐tolerance involves the continuous priming of Tregs by autoantigens, and in autoimmune disease suppression, the effector T‐cell response is continuously negated by potent disease‐specific Tregs that accumulate at the site of autoantigen presentation.

Role of Interleukin 23 Signaling in Clostridium difficile Colitis

Clostridium difficile is currently the leading cause of hospital-acquired infections in the United States. Here, we observed increased interleukin 23 (IL-23) protein levels in human colon biopsy specimens positive for C. difficile toxins, compared with levels in negative controls (P = .008) We also investigated the role of IL-23 during C. difficile infection, using 2 distinct murine models. Mice lacking IL-23 signaling had a significant increase in survival (100% [12 mice]), compared with control mice (16.7%-50% [12 mice]). These data suggest a new potential drug target for human C. difficile treatment and indicate the first link between IL-23 and disease severity during murine infection.

Requirements of NK Cells and Proinflammatory Cytokines in T Cell-Dependent Neonatal Autoimmune Ovarian Disease Triggered by Immune Complex

A model of neonatal autoimmune disease has been described recently in which an epitope-specific autoantibody to murine zona pellucida 3 induces severe ovarian disease in neonatal, but not adult, mice (neonatal AOD). The autoantibody forms immune complex with endogenous ovarian zona pellucida 3, and a pathogenic CD4+ T cell response is triggered. The basis for the predominant neonatal susceptibility has not been clarified. In this study innate immunity, including neonatal NK cells, in neonatal AOD was investigated. Neonatal spleen contained readily detectable NK1.1+TCRVβ−, but not NK1.1+TCRVβ+, cells. Ab depletion of NK1.1+TCRVβ− cells inhibited neonatal AOD development. Moreover, in adoptive transfer of neonatal AOD, recipient disease was ameliorated when either donor or recipient NK cells were depleted. Thus, NK cells operate in both induction and effector phases of the disease. IFN-γ was produced by neonatal NK cells in vivo, and it may be important in neonatal AOD. Indeed, ovaries with neonatal AOD expressed high levels of IFN-γ and TNF-α which correlated with disease severity, and the disease was inhibited by IFN-γ or TNF-α Ab. Importantly, disease was enhanced by recombinant IFN-γ, and treatment of T cell donors with IFN-γ Ab also significantly reduced adoptive transfer of neonatal AOD. Finally, neonatal AOD was ameliorated in mice deficient in FcγRIII and was enhanced in FcγRIIB-deficient mice. We conclude that neonatal NK cells promote pathogenic T cell response at multiple stages during neonatal autoimmune disease pathogenesis. Also operative in neonatal AOD are other mediators of the innate system, including proinflammatory cytokines and FcγRIII signaling.

Role of Leptin-Mediated Colonic Inflammation in Defense against Clostridium difficile Colitis

ABSTRACT The role of leptin in the mucosal immune response to Clostridium difficile colitis, a leading cause of nosocomial infection, was studied in humans and in a murine model. Previously, a mutation in the receptor for leptin (LEPR) was shown to be associated with susceptibility to infectious colitis and liver abscess due to Entamoeba histolytica as well as to bacterial peritonitis. Here we discovered that European Americans homozygous for the same LEPR Q223R mutation (rs1137101), known to result in decreased STAT3 signaling, were at increased risk of C. difficile infection (odds ratio, 3.03; P = 0.015). The mechanism of increased susceptibility was studied in a murine model. Mice lacking a functional leptin receptor (db/db) had decreased clearance of C. difficile from the gut lumen and diminished inflammation. Mutation of tyrosine 1138 in the intracellular domain of LepRb that mediates signaling through the STAT3/SOCS3 pathway also resulted in decreased mucosal chemokine and cell recruitment. Collectively, these data support a protective mucosal immune function for leptin in C. difficile colitis partially mediated by a leptin-STAT3 inflammatory pathway that is defective in the LEPR Q223R mutation. Identification of the role of leptin in protection from C. difficile offers the potential for host-directed therapy and demonstrates a connection between metabolism and immunity.

A Novel Near-Infrared Fluorescence Imaging Probe for In Vivo Neutrophil Tracking

The development and validation of a multiscopic near-infrared fluorescence (NIRF) probe, cinnamoyl-F-(D)L-F-(D)L-F-PEG-cyanine7 (cFlFlF-PEG-Cy7), that targets formyl peptide receptor on neutrophils using a mice ear inflammation model is described. Acute inflammation was induced in mice by topical application of phorbol-12-myristate-13-acetate to left ears 24 hours before probe administration. Noninvasive NIRF imaging was longitudinally performed up to 24 hours following probe injection. The in vivo neutrophil-targeting specificity of the probe was characterized by a blocking study with preadministration of excess nonfluorescent peptide cFlFlF-PEG and by an imaging study with a scrambled peptide probe cLFFFL-PEG-Cy7. NIRF imaging of mice injected with cinnamoyl-L-F-F-F-L-PEG-cyanine7 (cFlFlF-PEG-Cy7) revealed that the fluorescence intensity for inflamed left ears was approximately fourfold higher than that of control right ears at 24 hours postinjection. In comparison, the ratios acquired with the scrambled probe and from the blocking study were 1.5- and 2-fold at 24 hours postinjection, respectively. Moreover, a microscopic immunohistologic study confirmed that the NIRF signal of cFlFlF-PEG-Cy7 was associated with activated neutrophils in the inflammatory tissue. With this probe, in vivo neutrophil chemotaxis could be correlatively imaged macroscopically in live animals and microscopically at tissue and cellular levels.

Egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance

Autoimmune responses to meiotic germ cell antigens (MGCA) that are expressed on sperm and testis occur in human infertility and after vasectomy. Many MGCA are also expressed as cancer/testis antigens (CTA) in human cancers, but the tolerance status of MGCA has not been investigated. MGCA are considered to be uniformly immunogenic and nontolerogenic, and the prevailing view posits that MGCA are sequestered behind the Sertoli cell barrier in seminiferous tubules. Here, we have shown that only some murine MGCA are sequestered. Nonsequestered MCGA (NS-MGCA) egressed from normal tubules, as evidenced by their ability to interact with systemically injected antibodies and form localized immune complexes outside the Sertoli cell barrier. NS-MGCA derived from cell fragments that were discarded by spermatids during spermiation. They egressed as cargo in residual bodies and maintained Treg-dependent physiological tolerance. In contrast, sequestered MGCA (S-MGCA) were undetectable in residual bodies and were nontolerogenic. Unlike postvasectomy autoantibodies, which have been shown to mainly target S-MGCA, autoantibodies produced by normal mice with transient Treg depletion that developed autoimmune orchitis exclusively targeted NS-MGCA. We conclude that spermiation, a physiological checkpoint in spermatogenesis, determines the egress and tolerogenicity of MGCA. Our findings will affect target antigen selection in testis and sperm autoimmunity and the immune responses to CTA in male cancer patients.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1β, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

The acrosomal protein SP-10 (Acrv1) is an ideal marker for staging of the cycle of seminiferous epithelium in the mouse.

The study of spermatogenesis requires accurate identification of the stages of the cycle of the seminiferous epithelium. A stage refers to the unique association of germ cell types at a particular phase of development, as seen in a cross‐sectioned seminiferous tubule. Stage‐identification, however, is a daunting task. There are 12 stages represented in the mouse seminiferous epithelium. Stages are typically identified on the basis of the morphology of the developing acrosome of spermatids. Although the characteristic features of the acrosome are well‐documented in ultrastructure images, a reagent that can highlight the subtle differences in acrosome shape under the light microscope is lacking. Here we demonstrate that a polyclonal antibody raised against the mouse acrosomal protein SP‐10 is extremely useful for stage identification. Immunohistochemistry showed that the anti‐SP‐10 antibody is highly specific for the acrosome of spermatids, as no other cell type in the epithelium showed immunoreactivity. At lower magnification, the gross shape of the acrosome and the increasing intensity of immunostaining served as a guide for the identification of stages I–XII. At higher magnification, characteristic morphological features–such as whether the part of the acrosome that contacts the nuclear surface is round (stage III) or flat (stage IV) or curved (stage VI)–could be identified unambiguously. Overall, we present evidence that SP‐10 is a useful marker for staging the cycle of the seminiferous epithelium. The anti‐SP‐10 antibody works well in different fixatives, on paraffin‐embedded as well as cryosections, and has been shown to be useful for characterizing spermatogenic defects in mutant mice. Mol. Reprod. Dev. 81: 896–907, 2014.

Immunolocalization of TAR DNA-binding protein of 43 kDa (TDP-43) in mouse seminiferous epithelium

TAR DNA‐binding protein of 43 kDa (TDP‐43) is an evolutionarily conserved, ubiquitously expressed, multi‐functional DNA/RNA‐binding protein with roles in gene transcription, mRNA splicing, stability, transport, micro RNA biogenesis, and suppression of transposons. Aberrant expression of TDP‐43 in testis and sperm was recently shown to be associated with male infertility, which highlights the need to understand better the expression of TDP‐43 in the testis. We previously cloned TDP‐43 from a mouse testis cDNA library, and showed that it functions as a transcriptional repressor and regulates the precise spatiotemporal expression of the Acrv1 gene, which encodes the acrosomal protein SP‐10, during spermatogenesis. Here, we performed immunoblotting and immunohistochemistry of the mouse testis using four separate antibodies recognizing the amino and carboxyl termini of TDP‐43. TDP‐43 is present in the nuclei of germ cells as well as Sertoli cells. TDP‐43 expression begins in type B/intermediate spermatogonia, peaks in preleptotene spermatocytes, and becomes undetectable in leptotene and zygotene spermatocytes. Pachytene spermatocytes and early round spermatids again express TDP‐43, but its abundance diminishes later in spermatids (at steps 5–8). Interestingly, two of the four antibodies showed TDP‐43 expression in spermatids at steps 9–10, which coincides with the initial phase of the histone‐to‐protamine transition. Immunoreactivity patterns observed in the study suggest that TDP‐43 assumes different conformational states at different stages of spermatogenesis. TDP‐43 pathology has been extensively studied in the context of neurodegenerative diseases; its role in spermatogenesis warrants further detailed investigation of the involvement of TDP‐43 in male infertility.