PET/CT targeted tissue sampling reveals virus specific dIgA can alter the distribution and localization of HIV after rectal exposure.

Human immunodeficiency virus (HIV) vaccines have not been successful in clinical trials. Dimeric IgA (dIgA) in the form of secretory IgA is the most abundant antibody class in mucosal tissues, making dIgA a prime candidate for potential HIV vaccines. We coupled Positron Emission Tomography (PET) imaging and fluorescent microscopy of 64Cu-labeled, photoactivatable-GFP HIV (PA-GFP-BaL) and fluorescently labeled dIgA to determine how dIgA antibodies influence virus interaction with mucosal barriers and viral penetration in colorectal tissue. Our results show that HIV virions rapidly disseminate throughout the colon two hours after exposure. The presence of dIgA resulted in an increase in virions and penetration depth in the transverse colon. Moreover, virions were found in the mesenteric lymph nodes two hours after viral exposure, and the presence of dIgA led to an increase in virions in mesenteric lymph nodes. Taken together, these technologies enable in vivo and in situ visualization of antibody-virus interactions and detailed investigations of early events in HIV infection.

Localization of infection in neonatal rhesus macaques after oral viral challenge.

Vertical transmission of human immunodeficiency virus (HIV) can occur in utero, during delivery, and through breastfeeding. We utilized Positron Emission Tomography (PET) imaging coupled with fluorescent microscopy of 64Cu-labeled photoactivatable-GFP-HIV (PA-GFP-BaL) to determine how HIV virions distribute and localize in neonatal rhesus macaques two and four hours after oral viral challenge. Our results show that by four hours after oral viral exposure, HIV virions localize to and penetrate the rectal mucosa. We also used a dual viral challenge with a non-replicative viral vector and a replication competent SHIV-1157ipd3N4 to examine viral transduction and dissemination at 96 hours. Our data show that while SHIV-1157ipd3N4 infection can be found in the oral cavity and upper gastrointestinal (GI) tract, the small and large intestine contained the largest number of infected cells. Moreover, we found that T cells were the biggest population of infected immune cells. Thus, thanks to these novel technologies, we are able to visualize and delineate of viral distribution and infection throughout the entire neonatal GI tract during acute viral infection.

CCR2+ Ly6C(hi) inflammatory monocyte recruitment exacerbates acute disability following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating type of stroke that lacks a specific treatment. An intense immune response develops after ICH, which contributes to neuronal injury, disability, and death. However, the specific mediators of inflammation-induced injury remain unclear. The objective of the present study was to determine whether blood-derived CCR2+ Ly6C(hi) inflammatory monocytes contribute to disability. ICH was induced in mice and the resulting inflammatory response was quantified using flow cytometry, confocal microscopy, and neurobehavioral testing. Importantly, blood-derived monocytes were distinguished from resident microglia by differential CD45 staining and by using bone marrow chimeras with fluorescent leukocytes. After ICH, blood-derived CCR2+ Ly6C(hi) inflammatory monocytes trafficked into the brain, outnumbered other leukocytes, and produced tumor necrosis factor. Ccr2(-/-) mice, which have few circulating inflammatory monocytes, exhibited better motor function following ICH than control mice. Chimeric mice with wild-type CNS cells and Ccr2(-/-) hematopoietic cells also exhibited early improvement in motor function, as did wild-type mice after inflammatory monocyte depletion. These findings suggest that blood-derived inflammatory monocytes contribute to acute neurological disability. To determine the translational relevance of our experimental findings, we examined CCL2, the principle ligand for the CCR2 receptor, in ICH patients. Serum samples from 85 patients were collected prospectively at two hospitals. In patients, higher CCL2 levels at 24 h were independently associated with poor functional outcome at day 7 after adjusting for potential confounding variables. Together, these findings suggest that inflammatory monocytes worsen early disability after murine ICH and may represent a therapeutic target for patients.

CD134/CD137 dual costimulation-elicited IFN-γ maximizes effector T-cell function but limits Treg expansion.

T-cell tolerance to tumor antigens represents a major hurdle in generating tumor immunity. Combined administration of agonistic monoclonal antibodies (mAbs) to the costimulatory receptors CD134 plus CD137 can program T-cells responding to tolerogenic antigen to undergo expansion, and effector T-cell differentiation, and also elicits tumor immunity. Nevertheless, CD134 and CD137 agonists can also engage inhibitory immune components. To understand how immune stimulatory versus inhibitory components are regulated during CD134 plus CD137 dual costimulation (DCo), the current study utilized a model where DCo programs T-cells encountering a highly tolerogenic self-antigen to undergo effector differentiation. IFN-γ was found to have a pivotal role in maximizing the function of effector T-cells, while simultaneously limiting the expansion of CD4(+)CD25(+)Foxp3(+) Tregs. In antigen-responding effector T-cells, IFN-γ operates via a direct cell-intrinsic mechanism to cooperate with IL-2 to program maximal expression of granzyme B. Simultaneously, IFN-γ limits expression of the IL-2 receptor alpha chain (CD25) and IL-2 signaling through a mechanism that does not involve T-bet-mediated repression of IL-2. IFN-γ also limited CD25 and Foxp3 expression on bystanding CD4(+)Foxp3(+) Tregs, and limited the potential of these Tregs to expand. These effects could not be explained by the ability of IFN-γ to limit IL-2 availability. Taken together, during DCo IFN-γ interacts with IL-2 through distinct mechanisms to program maximal expression of effector molecules in antigen-responding T-cells, while simultaneously limiting Treg expansion.

Microglial responses after ischemic stroke and intracerebral hemorrhage.

Stroke is a leading cause of death worldwide. Ischemic stroke is caused by blockage of blood vessels in the brain leading to tissue death, while intracerebral hemorrhage (ICH) occurs when a blood vessel ruptures, exposing the brain to blood components. Both are associated with glial toxicity and neuroinflammation. Microglia, as the resident immune cells of the central nervous system (CNS), continually sample the environment for signs of injury and infection. Under homeostatic conditions, they have a ramified morphology and phagocytose debris. After stroke, microglia become activated, obtain an amoeboid morphology, and release inflammatory cytokines (the M1 phenotype). However, microglia can also be alternatively activated, performing crucial roles in limiting inflammation and phagocytosing tissue debris (the M2 phenotype). In rodent models, microglial activation occurs very early after stroke and ICH; however, their specific roles in injury and repair remain unclear. This review summarizes the literature on microglial responses after ischemic stroke and ICH, highlighting the mediators of microglial activation and potential therapeutic targets for each condition.

TGF-ß1 modulates microglial phenotype and promotes recovery after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results from the rupture of a blood vessel in the brain, leading to a mass of blood within the brain parenchyma. The injury causes a rapid inflammatory reaction that includes activation of the tissue-resident microglia and recruitment of blood-derived macrophages and other leukocytes. In this work, we investigated the specific responses of microglia following ICH with the aim of identifying pathways that may aid in recovery after brain injury. We used longitudinal transcriptional profiling of microglia in a murine model to determine the phenotype of microglia during the acute and resolution phases of ICH in vivo and found increases in TGF-ß1 pathway activation during the resolution phase. We then confirmed that TGF-ß1 treatment modulated inflammatory profiles of microglia in vitro. Moreover, TGF-ß1 treatment following ICH decreased microglial Il6 gene expression in vivo and improved functional outcomes in the murine model. Finally, we observed that patients with early increases in plasma TGF-ß1 concentrations had better outcomes 90 days after ICH, confirming the role of TGF-ß1 in functional recovery from ICH. Taken together, our data show that TGF-ß1 modulates microglia-mediated neuroinflammation after ICH and promotes functional recovery, suggesting that TGF-ß1 may be a therapeutic target for acute brain injury.

CX3CR1 signaling on monocytes is dispensable after intracerebral hemorrhage.

Intracerebral hemorrhage is a subset of stroke for which there is no specific treatment. The Ly6Chi CCR2+ monocytes have been shown to contribute to acute injury after intracerebral hemorrhage. The other murine monocyte subset expresses CX3CR1 and lower Ly6C levels, and contributes to repair in other disease models. We hypothesized that the Ly6Clo CX3CR1+ monocytes would contribute to recovery after intracerebral hemorrhage. Intracerebral hemorrhage was modeled by blood injection in WT and CX3CR1-null bone marrow chimeras. Neurological outcomes and leukocyte recruitment were quantified at various time points. Functional outcomes were equal at 1, 3, 7, and 14 days after intracerebral hemorrhage in both genotypes. No differences were observed in leukocyte recruitment between genotypes on either 3 or 7 days after intracerebral hemorrhage. A few hundred Ly6Clo monocytes were found in the ipsilateral hemisphere in each genotype and they did not change over time. Peripherally derived CX3CR1+ monocytes were observed in the perihematomal brain 7 and 14 days after intracerebral hemorrhage. Our data suggests CX3CR1 signaling on monocytes does not play an influential role in acute injury or functional recovery after intracerebral hemorrhage and therefore CX3CR1 is not a therapeutic target to improve outcome after intracerebral hemorrhage.