Pranita P. Sarangi

Regulatory T cells in virus infections

Summary:  This review discusses situations when the magnitude and function of immune responses to virus infection are influenced by regulatory T cells (Tregs). The focus is on CD4+CD25+ forkhead box protein 3+ natural Tregs (nTregs). The immune response may be limited in magnitude and efficacy when animals with normal nTreg function are infected with virus. This limitation can be observed both in vitro and in vivo. In the case of herpes simplex virus (HSV), animals depleted of nTregs prior to infection more effectively control the virus. With some virus infections, Treg responses (either nTregs or interleukin‐10‐dependent adaptive Tregs) appear to contribute to immune dysfunction, accounting for viral persistence and chronic tissue damage. This may occur with hepatitis C virus and some retrovirus infections that include human immunodeficiency virus (HIV). Under other circumstances, the nTreg response is judged to be beneficial, as it may help limit the severity of tissue damage associated with an immunoinflammatory reaction to virus infection. Such a situation occurs in HSV‐induced immunopathological lesions in the eye. With HIV, nTregs may help limit chronic immune activation that may precede collapse of the immune system. This review also discusses how virus infections become recognized by nTreg responses and how such responses might be manipulated to increase immunity or to limit virus‐induced immunopathology.

Recombinant human activated protein C inhibits integrin-mediated neutrophil migration

Integrin-mediated cell migration is central to many biologic and pathologic processes. During inflammation, tissue injury results from excessive infiltration and sequestration of activated leukocytes. Recombinant human activated protein C (rhAPC) has been shown to protect patients with severe sepsis, although the mechanism underlying this protective effect remains unclear. Here, we show that rhAPC directly binds to beta(1) and beta(3) integrins and inhibits neutrophil migration, both in vitro and in vivo. We found that human APC possesses an Arg-Gly-Asp (RGD) sequence, which is critical for the inhibition. Mutation of this sequence abolished both integrin binding and inhibition of neutrophil migration. In addition, treatment of septic mice with a RGD peptide recapitulated the beneficial effects of rhAPC on survival. Thus, we conclude that leukocyte integrins are novel cellular receptors for rhAPC and the interaction decreases neutrophil recruitment into tissues, providing a potential mechanism by which rhAPC may protect against sepsis.

Innate Recognition Network Driving Herpes Simplex Virus-Induced Corneal Immunopathology: Role of the Toll Pathway in Early Inflammatory Events in Stromal Keratitis

ABSTRACT Ocular infection with herpes simplex virus (HSV) sets off an array of events that succeed in clearing virus from the cornea but leaves the tissue with a CD4+ T-cell-orchestrated chronic inflammatory lesion that impairs vision. We demonstrate that Toll-like receptor (TLR) signaling forms a part of the recognition system that induces the syndrome that eventually culminates in immunopathology. Accordingly, in a comparison of the outcomes of infection in wild-type (WT) mice and those lacking TLR function, it was apparent that the absence of TLR2 and, to a lesser extent, TLR9 resulted in significantly diminished lesions. Similarly, mice lacking the adapter molecule MyD88 were resistant to lesion development, but such animals were also unable to control infection, with most succumbing to lethal encephalitis. The susceptibility of TLR4−/− animals was also evaluated. These animals developed lesions, which were more severe, more rapidly than did WT animals. We discuss the possible mechanisms by which early recognition of HSV constituents impacts the subsequent development of immunopathological lesions.

Uropod elongation is a common final step in leukocyte extravasation through inflamed vessels

Uropod elongation occurs during leukocyte extravasation.

In Vivo Kinetics of GITR and GITR Ligand Expression and Their Functional Significance in Regulating Viral Immunopathology

ABSTRACT This report evaluates the role of interaction between glucocorticoid-induced tumor necrosis factor receptor (GITR) and GITR ligand (GITR-L) in the immunoinflammatory response to infection with herpes simplex virus (HSV). Both GITR and GITR-L were transiently upregulated after ocular HSV infection, on antigen-specific T cells and antigen-presenting cells, respectively, in the draining lymph node (DLN). In addition, virus-specific T-cell responses in the DLN and spleen were enhanced by anti-GITR antibody treatment, an outcome expected to result in more severe inflammatory lesions. Intriguingly, the treatment resulted in significantly diminished T-cell-mediated ocular lesions. The explanation for these findings was that anti-GITR antibody treatment caused a reduced production of ocular MMP-9, a molecule involved in ocular angiogenesis, an essential step in the pathogenesis of herpetic keratitis. Our results are the first observations to determine in vivo kinetics of GITR and GITR-L expression after virus infection, and they emphasize the role of GITR-GITR-L interaction to regulate virus-induced immunoinflammatory lesions.

IL-10 and Natural Regulatory T Cells: Two Independent Anti-Inflammatory Mechanisms in Herpes Simplex Virus-Induced Ocular Immunopathology

Two prominent anti-inflammatory mechanisms involved in controlling HSV-1-induced corneal immunopathology (stromal keratitis or SK) are the production of the cytokine IL-10 and the activity of natural regulatory T cells (nTregs). It is not known whether, under in vivo conditions, IL-10 and nTregs influence the corneal pathology independently or in concert. In the current study using wild-type and IL-10−/− animals, we have assessed the activity of nTregs in the absence of IL-10 both under in vitro and in vivo conditions. The IL-10−/− animals depleted of nTregs before ocular infection showed more severe SK lesions as compared with the undepleted IL-10−/− animals. In addition, nTregs purified from naive WT and IL-10−/− animals were equally able to suppress the proliferation and the cytokine production from anti-CD3-stimulated CD4+CD25− T cells in vitro. Furthermore, intracellular cytokine staining results indicated that nonregulatory cells expressing B220 and CD25 markers were the major IL-10-producing cell types in the lymphoid tissues of HSV-infected mice. In contrast, in the infected corneas, cells with the CD11b+Gr1+ phenotype along with a minor population of Foxp3−CD4+ and a few F4/80+ cells produced IL-10. Our current investigations indicate that at least two independent anti-inflammatory mechanisms are involved in limiting the corneal lesions in SK, both of which may need to be modulated to control SK therapeutically.

In Vitro-Generated Antigen-Specific CD4+ CD25+ Foxp3+ Regulatory T Cells Control the Severity of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions

ABSTRACT Generating and using regulatory T cells (Tregs) to modulate inflammatory disease represents a valuable approach to therapy but has not yet been applied as a means to control virus-induced immunopathological reactions. In this report, we developed a simplified technique that used unfractionated splenocytes as a precursor population and showed that stimulation under optimized conditions for 5 days with solid-phase anti-CD3 monoclonal antibody in the presence of transforming growth factor β (TGF-β) and interleukin-2 could induce up to 90% of CD4+ T cells to become Foxp3+ and able to mediate suppression in vitro. CD11c+ dendritic cells were intricately involved in the conversion process and, once modified in the presence of TGF-β, could convert Foxp3− CD4+ cells into Foxp3+ CD4+cells by producing TGF-β. The converted cells had undergone cell division, and the majority of them expressed activation markers along with surface molecules that would facilitate their migration into tissue sites. The primary reason for our study was to determine if such in vitro-converted Tregs could be used in vivo to influence the outcome of a virus-induced immunoinflammatory lesion in the eye caused by herpes simplex virus infection. We could show in three separate models of herpetic stromal keratitis that adoptive transfers of in vitro-converted Tregs effectively diminished lesion severity, especially when given in the initial phases of infection. The suppression effect in vivo appeared to be polyspecific. The protocol we have developed could provide a useful additional approach to control virus-induced inflammatory disease.

Activated protein C action in inflammation.

Activated protein C (APC) is a natural anticoagulant that plays an important role in coagulation homeostasis by inactivating the procoagulation factor Va and VIIIa. In addition to its anticoagulation functions, APC also has cytoprotective effects such as anti‐inflammatory, anti‐apoptotic, and endothelial barrier protection. Recently, a recombinant form of human APC (rhAPC or drotrecogin alfa activated; known commercially as ‘Xigris’) was approved by the US Federal Drug Administration for treatment of severe sepsis associated with a high risk of mortality. Sepsis, also known as systemic inflammatory response syndrome (SIRS) resulting from infection, is a serious medical condition in critical care patients. In sepsis, hyperactive and dysregulated inflammatory responses lead to secretion of pro‐ and anti‐inflammatory cytokines, activation and migration of leucocytes, activation of coagulation, inhibition of fibrinolysis, and increased apoptosis. Although initial hypotheses focused on antithrombotic and profibrinolytic functions of APC in sepsis, other agents with more potent anticoagulation functions were not effective in treating severe sepsis. Furthermore, APC therapy is also associated with the risk of severe bleeding in treated patients. Therefore, the cytoprotective effects, rather than the anticoagulant effect of APC are postulated to be responsible for the therapeutic benefit of APC in the treatment of severe sepsis.

Enhanced viral immunoinflammatory lesions in mice lacking IL-23 responses

Herpes simplex virus (HSV) infection of the cornea culminates in an immunopathological lesion (stromal keratitis--SK) that impairs vision. This report shows that HSV infection results in IL-23 up-regulation, but if this response fails to occur, as was noted in p19-/- mice, the severity of lesions, their incidence and the level of viral induced angiogenesis were significantly increased compared to wild-type (WT) animals (p<0.05). The higher disease severity in p19-/- mice appeared to be the consequence of an increased IL-12 response that in turn led to the induction of higher numbers of IFN-gamma producing CD4(+)T cells, the principal orchestrators of SK. Our results indicate that the severity of HSV induced immunopathological lesions may be mainly the consequence of IL-12 driven Th1 T cell reactions rather than the action of IL-17 producing cells controlled by IL-23.

Depletion of MCP-1 increases development of herpetic stromal keratitis by innate immune modulation.

Chemokines are important chemoattractant inflammatory molecules, but their interdependent network in disease pathogenesis remains unclear. Studies in mouse models have shown that herpetic stromal keratitis (SK) is produced by the consequence of a tissue‐destructive immunoinflammatory reaction involving herpes simplex virus type 1 (HSV) infection. Here we found that ocular HSV infection leads to increased expression of monocyte chemoattractant protein‐1 (MCP‐1), one of the major chemoattractants for immune cells that express CCR2, in the SK cornea. However, MCP‐1 is unlikely to be a chemoattractant for infiltrating Gr‐1+, CD11b+ cells in SK, as these cells are found to be CCR2 negative. Nevertheless, infection of MCP‐1−/− mice resulted in more severe SK lesion severity compared with WT mice (P<0.01). We demonstrated that the loss of MCP‐1 in the SK cornea caused a significant overexpression of macrophage inflammatory protein‐2 (MIP‐2) (P<0.01) on days 2 and 4 postinfection and increased infiltration of inflammatory cells (Gr‐1‐high and CD11b+) expressing CXCR2, a receptor for MIP‐2, into the cornea. Subsequently, increased infiltration of inflammatory cells accelerated by MIP‐2 overexpression might result in the high production of inflammatory molecules, including vascular endothelial growth factor (VEGF) and IL‐1β in SK, as well as CpG oligodeoxynucleotide (ODN)‐implanted eyes of MCP‐1−/− mice. These results indicate that MCP‐1 in the SK cornea might regulate the expression of other chemokines, as well as the infiltration of inflammatory cells and control development of SK.