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Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy.

Martinez-Skinner AL, Araínga MA, Puligujja P, Palandri DL, Baldridge HM, Edagwa BJ, McMillan JM, Mosley RL, Gendelman HE - PLoS ONE (2015)

Bottom Line: Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen.Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution.Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5880, United States of America.

ABSTRACT
Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.

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Identification of nanoATV within spleen and lymph node.Confocal microscopy images of cryosectioned spleen and mesenteric lymph nodes collected 12 h after intraperitoneal injection of CF633-labeled nanoATV (pink). Cell nuclei were stained with DAPI (blue); macrophages were stained with Alexa Fluor 488-linked F4/80 antibody (green). Immunostainings as follows: (A) H&E of spleen; (B) F4/80 labeled splenic macrophages; (C) CF633-nanoATV within spleen; (D) Overlay of (B) and (C); (E) F4/80 labeled splenic macrophages; (F) CF633-nanoATV in spleen; (G) overlay of (E) and (F); (H) H&E of lymph nodes; (I) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes; (J) CF633-nanoATV within lymph nodes; (K) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes. Abbreviations: RP (red pulp), MZ (marginal zone), F (B-cell rich), T (T-cell rich), M (medulla), and SCS (subcapsular sinus). Magnifications: 40X for B, C, D, J and K; 10X for A, E, F, G, H and I. (scale bars = 100 μm).
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pone.0145966.g005: Identification of nanoATV within spleen and lymph node.Confocal microscopy images of cryosectioned spleen and mesenteric lymph nodes collected 12 h after intraperitoneal injection of CF633-labeled nanoATV (pink). Cell nuclei were stained with DAPI (blue); macrophages were stained with Alexa Fluor 488-linked F4/80 antibody (green). Immunostainings as follows: (A) H&E of spleen; (B) F4/80 labeled splenic macrophages; (C) CF633-nanoATV within spleen; (D) Overlay of (B) and (C); (E) F4/80 labeled splenic macrophages; (F) CF633-nanoATV in spleen; (G) overlay of (E) and (F); (H) H&E of lymph nodes; (I) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes; (J) CF633-nanoATV within lymph nodes; (K) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes. Abbreviations: RP (red pulp), MZ (marginal zone), F (B-cell rich), T (T-cell rich), M (medulla), and SCS (subcapsular sinus). Magnifications: 40X for B, C, D, J and K; 10X for A, E, F, G, H and I. (scale bars = 100 μm).

Mentions: To determine the cellular distribution of nanoATV within tissues, CF633-labeled nanoATV was administered to mice and specific cell markers were used to determine cell depots in liver, spleen, and lymph nodes using confocal microscopy. We chose to assess this at 12 h post injection as that was the time when maximal drug concentrations were detected in these tissues (Fig 3). Cryosections of liver were immunostained for the Kupffer cell marker F4/80 (Fig 4). Individual panels show nuclei (blue, Fig 4A), F4/80+ Kupffer cells (green, Fig 4B), nanoATV alone (pink, Fig 4C), and the merged image (Fig 4D) and demonstrate the presence of nanoATV (pink) in Kupffer cells (F4/80 positive, green). Drug particles were also observed in the medullary sinus of mesenteric lymph nodes and within the spleen along follicular borders (Fig 5). Fig 5B and 5E show F4/80+ stained splenic macrophages (green) and Fig 5C and 5F show accumulation of nanoATV (pink) in spleen. The merged images (Fig 5D and 5G) demonstrate the presence of nanoATV along the borders of splenic follicles. Fig 5I and 5K show localization of nanoATV and F4/80+ cells in mesenteric lymph nodes. Localization of nanoATV was clearly visible in red pulp macrophages, marginal zone macrophages (Fig 5) and marginal metalophillic cells (Fig 6) along the marginal zone of the spleen.


Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy.

Martinez-Skinner AL, Araínga MA, Puligujja P, Palandri DL, Baldridge HM, Edagwa BJ, McMillan JM, Mosley RL, Gendelman HE - PLoS ONE (2015)

Identification of nanoATV within spleen and lymph node.Confocal microscopy images of cryosectioned spleen and mesenteric lymph nodes collected 12 h after intraperitoneal injection of CF633-labeled nanoATV (pink). Cell nuclei were stained with DAPI (blue); macrophages were stained with Alexa Fluor 488-linked F4/80 antibody (green). Immunostainings as follows: (A) H&E of spleen; (B) F4/80 labeled splenic macrophages; (C) CF633-nanoATV within spleen; (D) Overlay of (B) and (C); (E) F4/80 labeled splenic macrophages; (F) CF633-nanoATV in spleen; (G) overlay of (E) and (F); (H) H&E of lymph nodes; (I) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes; (J) CF633-nanoATV within lymph nodes; (K) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes. Abbreviations: RP (red pulp), MZ (marginal zone), F (B-cell rich), T (T-cell rich), M (medulla), and SCS (subcapsular sinus). Magnifications: 40X for B, C, D, J and K; 10X for A, E, F, G, H and I. (scale bars = 100 μm).
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Related In: Results  -  Collection

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pone.0145966.g005: Identification of nanoATV within spleen and lymph node.Confocal microscopy images of cryosectioned spleen and mesenteric lymph nodes collected 12 h after intraperitoneal injection of CF633-labeled nanoATV (pink). Cell nuclei were stained with DAPI (blue); macrophages were stained with Alexa Fluor 488-linked F4/80 antibody (green). Immunostainings as follows: (A) H&E of spleen; (B) F4/80 labeled splenic macrophages; (C) CF633-nanoATV within spleen; (D) Overlay of (B) and (C); (E) F4/80 labeled splenic macrophages; (F) CF633-nanoATV in spleen; (G) overlay of (E) and (F); (H) H&E of lymph nodes; (I) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes; (J) CF633-nanoATV within lymph nodes; (K) overlay of F4/80 labeled macrophages and CF633-nanoATV in lymph nodes. Abbreviations: RP (red pulp), MZ (marginal zone), F (B-cell rich), T (T-cell rich), M (medulla), and SCS (subcapsular sinus). Magnifications: 40X for B, C, D, J and K; 10X for A, E, F, G, H and I. (scale bars = 100 μm).
Mentions: To determine the cellular distribution of nanoATV within tissues, CF633-labeled nanoATV was administered to mice and specific cell markers were used to determine cell depots in liver, spleen, and lymph nodes using confocal microscopy. We chose to assess this at 12 h post injection as that was the time when maximal drug concentrations were detected in these tissues (Fig 3). Cryosections of liver were immunostained for the Kupffer cell marker F4/80 (Fig 4). Individual panels show nuclei (blue, Fig 4A), F4/80+ Kupffer cells (green, Fig 4B), nanoATV alone (pink, Fig 4C), and the merged image (Fig 4D) and demonstrate the presence of nanoATV (pink) in Kupffer cells (F4/80 positive, green). Drug particles were also observed in the medullary sinus of mesenteric lymph nodes and within the spleen along follicular borders (Fig 5). Fig 5B and 5E show F4/80+ stained splenic macrophages (green) and Fig 5C and 5F show accumulation of nanoATV (pink) in spleen. The merged images (Fig 5D and 5G) demonstrate the presence of nanoATV along the borders of splenic follicles. Fig 5I and 5K show localization of nanoATV and F4/80+ cells in mesenteric lymph nodes. Localization of nanoATV was clearly visible in red pulp macrophages, marginal zone macrophages (Fig 5) and marginal metalophillic cells (Fig 6) along the marginal zone of the spleen.

Bottom Line: Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen.Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution.Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5880, United States of America.

ABSTRACT
Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.

Show MeSH
Related in: MedlinePlus