Daniel R. Barreda

Antimicrobial mechanisms of fish phagocytes and their role in host defense

Phagocytosis is a primitive defense mechanism in all multicellular animals. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens. These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, macrophages and neutrophils can be activated to produce a number of highly toxic molecules. Production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens. Studies in fish suggest that the biological basis of these inducible killing mechanisms is similar to those described in mammals. More recent work suggest novel roles for regulating these killing responses in fish. In this review, we describe the biological basis of these killing mechanisms and how they are regulated in fish.

Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death.

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).

Pivotal Advance: Peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells

Breaking the long‐held paradigm that primary B cells are not phagocytic, several studies have demonstrated recently that B cells from fish, amphibians, and reptilians have a significant phagocytic capacity. Whether such capacity has remained conserved in certain mammalian B cell subsets is presently an enigma. Here, we report a previously unrecognized ability of PerC B‐1a and B‐1b lymphocytes to phagocytose latex beads and bacteria. In contrast, B‐2 lymphocytes had an almost negligible ability to internalize these particles. Upon phagocytosis, B‐1a and B‐1b cells were able to mature their phagosomes into phagolysosomes and displayed the ability to kill internalized bacteria. Importantly, B‐1a and B‐1b cells effectively present antigen recovered from phagocytosed particles to CD4+ T cells. However, these cells showed a much lower competence to present soluble antigen or antigen from large, noninternalized particles. B‐1 B cells presented particulate and soluble antigen to CD4+ T cells more efficiently than macrophages, whereas DCs were the most potent APCs. The novel phagocytic and microbicidal abilities identified in B‐1 B lymphocytes strengthen the innate nature that has long been attributed to these cells. In the context of adaptive immunity, we show that these innate immune processes are relevant, as they enable B‐1 B cells to present phagocytosable particulate antigen. These capacities position these cells at the crossroads that link innate with adaptive immune processes. In a broader context, these newly identified capacities of B‐1 B cells further support the previously recognized functional, developmental, and evolutionary relationships between these cells and macrophages.

Production of a macrophage growth factor(s) by a goldfish macrophage cell line and macrophages derived from goldfish kidney leukocytes

We recently established a spontaneously proliferating macrophage cell line from the goldfish (GMCL), and in this report demonstrate the production of a macrophage-specific growth factor(s) (MGFs) by these cells. The supernatants from GMCL cultures induced proliferation and differentiation of macrophage-like cells from kidney hematopoietic tissues of goldfish. Kidney leukocytes cultured at 6.25 x 10(4)cells/ml in the presence of GMCL-derived MGFs proliferated during two weeks of cultivation, whereas those cultured without the MGFs did not. Leukocytes cultured at higher densities (2.5 x 10(5) cells/ml) proliferated in the absence of exogenous growth factor, but not to the same extent as those stimulated with GMCL-derived MGFs, suggesting that kidney leukocytes may produce endogenous MGFs. At higher cell density (1 x 10(6) cells/ml), kidney leukocytes multiplied extensively over a two-week cultivation period in the absence of exogenous GMCL-derived MGFs. The supernatants from these cultures restored the proliferative ability of leukocytes cultured at low densities, providing direct evidence of MGFs production by kidney leukocytes. The predominant cell-type in cultures grown in the presence of GMCL or kidney leukocyte-MGFs was the macrophage based on the following criteria: (1) non-specific esterase staining; (2) morphologic similarity to GMCL; (3) phagocytosis of the bacterium, A. salmonicida; (4) production of reactive oxygen and nitrogen intermediates in response to stimulation with macrophage activating factors and/or bacterial lipopolysaccharide; and (5) flow cytometric analyses. Both in vitro-derived kidney macrophage (IVDKM) and GMCL cultures contained three distinct populations of cells, (determined by flow cytometry), suggesting that these macrophage cultures are comprised of cells arrested at distinct differentiation junctures in macrophage development. Production of MGFs by macrophages and kidney leukocytes may play an important role in regulating macrophage hematopoiesis in fish.

Differential kinase requirements in human and mouse Fc‐gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcγRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcγR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src‐related tyrosine kinases (SRTKs) and phosphatidyl inositol 3‐kinases (PI3K) play important roles in FcγR phagocytosis. Herein, we demonstrate that these kinases are not required for FcγR endocytosis. Endocytosis of heat‐aggregated IgG (HA‐IgG) by COS‐1 cells stably transfected with FcγRIIA or chimeric FcγRI‐γ‐γ (EC‐TM‐CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG‐sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow‐derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcγRIIA was cross‐linked using HA‐IgG or EA. However, inhibition of FcγR phosphorylation prevented only FcγR phagocytosis. Finally, biochemical analyses of PI3K(p85)‐Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcγR phagocytosis and endocytosis. Overall, our results indicate that FcγR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition‐based model in which PI3K and c‐Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

Antimicrobial mechanisms of fish leukocytes.

Early activation and coordination of innate defenses are critical for effective responses against infiltrating pathogens. Rapid engagement of immune cells provides a critical first line of defense soon after pathogen infiltration. Activation leads to a well-orchestrated set of events that sees the induction and regulation of intracellular and extracellular antimicrobial defenses. An array of regulatory mediators, highly toxic soluble molecules, degradative enzymes and antimicrobial peptides provides maximal protection against a wide range of pathogens while limiting endogenous damage to host tissues. In this review we highlight recent advances in our understanding of innate cellular antimicrobial responses of teleost fish and discuss their implications to cell survival, immunomodulation and death. The evolutionary conservation of these responses is a testament to their effectiveness against pathogen infiltration and their commitment to effective maintenance of host homeostasis. Importantly, recent developments in teleost fish systems have identified novel host defense strategies that may be unique to this lower vertebrate group or may point to previously unknown innate mechanisms that also play a significant role in higher vertebrate host immunity.

Short-term feed restriction impairs the absorptive function of the reticulo-rumen and total tract barrier function in beef cattle1

The objective of this study was to evaluate whether different severities of short-term feed restriction (FR) affect the absorptive function of the reticulo-rumen and total tract barrier function in beef cattle. Eighteen ruminally cannulated and ovariectomized Angus × Hereford heifers were blocked by BW into 3 blocks, with blocks conducted sequentially. Treatments were imposed during the 5-d FR period by restricting heifers to 75 (FR75), 50 (FR50) or 25% (FR25) of the ad libitum feed intake measured during a 5-d baseline period (BASE) occurring immediately before FR. Throughout the study, heifers were housed in individual pens (9 m(2)) and were fed the same diet (60% forage:40% concentrate) with free access to water. Dry matter intake was measured daily and ruminal pH was measured every 2 min throughout the study. Ruminal fluid and blood samples were collected on d 3 of the BASE and FR periods, and the temporarily isolated and washed reticulo-rumen technique was used to evaluate short-chain fatty acid (SCFA) absorption on d 5 of the BASE and FR periods. Total tract barrier function was evaluated starting on d 2 of the BASE and FR periods using a pulse dose of Cr-EDTA followed by 48 h of total urine collection. Data were analyzed using the Proc Mixed procedure of SAS with the fixed effects of block, treatment, period, and the treatment × period interaction, the random effect of cow nested in block with period included as a repeated measure. Dry matter intake did not differ among treatments during BASE but, as imposed by the experimental model, DMI during FR relative to BASE equated to 70, 49, and 25%, which was close to the targeted values of 75, 50, and 25% (treatment × period, P < 0.001). A treatment × period interaction (P < 0.001) was also detected for ruminal SCFA concentration with the concentration decreasing as the severity of FR increased, whereas there were no differences during BASE. Mean ruminal pH increased during FR with increasing severity of FR, but was not different during BASE (treatment × period, P < 0.001). Absorption of SCFA across the reticulo-rumen tended to decrease with increasing severity of FR (P = 0.08). For individual SCFA, acetate absorption (mmol/h) tended (P = 0.057) to be less for FR25 and FR50 when compared with FR75 and decreased (P = 0.05) by almost 70 mmol/h at FR25 and FR50 relative to BASE (322mmol/h). Heifers restricted to 25% (FR25) feed had greater urinary Cr recovery during FR than BASE, whereas no changes were detected for FR75 and FR50. This study indicates that moderate severities of short-term FR decrease the absorptive function of the reticulo-rumen, but more severe FR is required to compromise total tract barrier function in beef cattle.

Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.

The advent of flow cytometry-based applications has significantly impacted the study of cellular apoptosis. Propidium iodide (PI) is a commonly used viability stain in these studies. Unfortunately, we find that conventional Annexin V/PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Both primary cells and cell lines are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. This distribution spans a wide range of animal models including mice, swine, avian, and teleost fish and potentially affects up to 1016 out of 1019 of peer-reviewed papers published in this area since 1995. We show that the primary ramifications from these findings relate to cells experiencing changes in RNA content. Virally infected cells, for example, are qualified as undergoing apoptosis in response to infection based on conventional staining protocols; in fact, these cells are alive and actively producing viral RNA that can serve to produce additional infectious viral particles. Based on our observations we propose a modified protocol, show that it overcomes previous drawbacks for this technique, and that it will allow for more accurate assessment of cell death across various platforms.

Macrophage activation differentially modulates particle binding, phagocytosis and downstream antimicrobial mechanisms.

Phagocytosis provides a critical first line of defense against invading pathogens. Engagement of particles through receptor-mediated binding precedes internalization and induction of cellular antimicrobial responses. Phagocytes have the capacity to differentially regulate binding and internalization processes through changes in their receptor profile and modulation of downstream events. This is necessary for the intricate control of phagocytic antimicrobial responses. Several methods are available for evaluation of phagocytosis. Unfortunately, none allow for accurate quantitation of both binding and internalization events. To overcome these limitations, we have developed a novel phagocytosis assay based on a multi-spectral imaging flow cytometry platform. This assay discriminates between internalized and surface-bound particles in a statistically robust manner and allows multi-parametric analysis of phagocytosis and downstream anti-microbial responses. We also devised a novel approach for examination of phagolysosome fusion, which provides an improved capacity for quantitative assessment of phagolysosome fusion in mixed populations of intact cells. Importantly, our approaches are likely amenable to a broad range of comparative model systems based on our examination of murine RAW 264.7 cells and a goldfish primary kidney macrophage (PKM) model system. The latter allowed us to examine the evolutionary conservation of phagocytic antimicrobial responses in a lower vertebrate model. While it has been previously reported that mixed populations of these macrophage cultures are phagocytic, it remained unclear if sub-populations within them contributed differentially to this activity. In accordance with higher vertebrate models, we found that differentiation along the macrophage pathway leads to an increased capacity for phagocytosis in goldfish PKM. Interestingly, cellular activation differentially regulated particle internalization in PKM monocyte and mature macrophage subsets. We also found differential regulation of phagolysosome fusion and downstream production of reactive oxygen intermediates (ROI). The temporal activation of specific phagocytic antimicrobial responses at distinct stages of PKM differentiation suggests specialization within the macrophage compartment early in evolution, geared to meet specific host immunity requirements within specialized niches.

The effects of feeding 3-nitrooxypropanol on methane emissions and productivity of Holstein cows in mid lactation.

The objective of the current study was to determine the effects of adding 3-nitrooxypropanol to the diet of lactating Holstein cows on methane emissions, rumen fermentation, ruminal microbial profile, and milk production. Twelve ruminally cannulated Holstein cows in midlactation were used in a crossover design study with 28-d periods. Cows were fed a diet containing 38% forage on a dry matter basis with either 2,500 mg/d of 3-nitrooxypropanol (fed as 25 g of 10% 3-nitrooxypropanol on silicon dioxide) or 25 g/d of silicon dioxide (control). After a 21-d diet adaptation period, dry matter intake (DMI) and milk yield were recorded daily. Rumen fluid and digesta were collected on d 22 and 28 for volatile fatty acid analysis and microbial profiling. Enteric methane emissions were measured on d 23 to 27 using the sulfur hexafluoride tracer gas technique. Feeding 3-nitrooxypropanol did not affect DMI; however, methane production was reduced from 17.8 to 7.18 g/kg of DMI. No change in milk or milk component yields was observed, but cows fed 3-nitrooxypropanol gained more body weight than control cows (1.06 vs. 0.39 kg/d). Concentrations of total volatile fatty acids in ruminal fluid were not affected by treatment, but a reduction in acetate proportion and a tendency for an increase in propionate proportion was noted. As such, a reduction in the acetate-to-propionate ratio was observed (2.02 vs. 2.36). Protozoa counts were not affected by treatment; however, a reduction in methanogen copy count number was observed when 3-nitrooxypropanol was fed (0.95 vs. 2.69 × 10(8)/g of rumen digesta). The data showed that feeding 3-nitrooxypropanol to lactating dairy cows at 2,500 mg/d can reduce methane emissions without compromising DMI or milk production.