Martin P. Hosking

Zinc sequestration by the neutrophil protein calprotectin enhances Salmonella growth in the inflamed gut

Neutrophils are innate immune cells that counter pathogens by many mechanisms, including release of antimicrobial proteins such as calprotectin to inhibit bacterial growth. Calprotectin sequesters essential micronutrient metals such as zinc, thereby limiting their availability to microbes, a process termed nutritional immunity. We find that while calprotectin is induced by neutrophils during infection with the gut pathogen Salmonella Typhimurium, calprotectin-mediated metal sequestration does not inhibit S. Typhimurium proliferation. Remarkably, S. Typhimurium overcomes calprotectin-mediated zinc chelation by expressing a high affinity zinc transporter (ZnuABC). A S. Typhimurium znuA mutant impaired for growth in the inflamed gut was rescued in the absence of calprotectin. ZnuABC was also required to promote the growth of S. Typhimurium over that of competing commensal bacteria. Thus, our findings indicate that Salmonella thrives in the inflamed gut by overcoming the zinc sequestration of calprotectin and highlight the importance of zinc acquisition in bacterial intestinal colonization.

G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker.

Differential roles for CXCR3 in CD4+ and CD8+ T cell trafficking following viral infection of the CNS

Lymphocyte infiltration into the central nervous system (CNS) following viral infection represents an important component of host defense and is required for control of viral replication. However, the mechanisms governing inflammation in response to viral infection of the CNS are not well understood. Following intracranial (i.c.) infection of susceptible mice with mouse hepatitis virus (MHV), mice develop an acute encephalomyelitis followed by a chronic demyelinating disease. The CXC chemokine ligand 10 (CXCL10) is expressed following MHV infection and signals T cells to migrate into the CNS. The functional contribution of the CXCL10 receptor CXCR3 in host defense and disease in response to MHV infection was evaluated. The majority of CD4+ and CD8+ T cells infiltrating the CNS following MHV infection express CXCR3. Administration of anti‐CXCR3 antibody reduced CD4+ T cell infiltration (p⩽0.05), while CD8+ T cell trafficking was not affected. Anti‐CXCR3 treatment during chronic disease correlated with improved motor skills and reduced demyelination. The selective effect of anti‐CXCR3 treatment on CD4+ T cells was not the result of either reduced proliferation or modulation in chemokine receptor gene expression. Therefore, CXCR3 signaling has a non‐redundant role in T cell subset trafficking in response to viral infection.

The Role of Chemokines during Viral Infection of the CNS

Viral infection of the central nervous system (CNS) poses unique challenges to the immune system with regards to controlling and eliminating the invading pathogen. These obstacles include the presence of a blood–brain barrier (BBB) that provides a physical and physiological barrier that is difficult for cells and molecules to cross, the absence of classic lymphatic drainage that may impair the generation of an adaptive immune response, and limited MHC class I or II expression on resident cells of the CNS, even during periods of neuroinflammation. In addition, the CNS is composed of a variety of highly specialized cells, many of which have limited renewal capacity, that represent potential targets of infection by numerous different viruses. Nonetheless, antigen-specific lymphocytes are ultimately able to accumulate within the CNS and contribute to defense by reducing or eliminating the invading viral pathogen. Alternatively, infiltration of activated cells of the immune system may be detrimental, as these cells can contribute to neuropathology that may result in long-term cellular damage or death. Understanding the mechanisms that govern leukocyte trafficking from the microvasculature into the CNS parenchyma is therefore critical for comprehending the molecular and cellular events that control neuroinflammation following infection by neurotropic viruses. Chemokines, small (8–10 kDa) proteins expressed by almost all nucleated cell types, are divided into four subfamilies based upon the number and spacing of conserved cysteine residues present within the amino terminus of the protein. Chemokine function is controlled through often promiscuous signaling via seven transmembrane G-protein-coupled receptors. While initially characterized as important in inflammation by targeting distinct leukocyte populations, chemokines are now considered critical mediators of a variety of biological processes, including development, tissue homeostasis, and coordinated immune responses during viral infection.

A Protective Role for ELR+ Chemokines during Acute Viral Encephalomyelitis

The functional role of ELR-positive CXC chemokines in host defense during acute viral-induced encephalomyelitis was determined. Inoculation of the neurotropic JHM strain of mouse hepatitis virus (JHMV) into the central nervous system (CNS) of mice resulted in the rapid mobilization of PMNs expressing the chemokine receptor CXCR2 into the blood. Migration of PMNs to the CNS coincided with increased expression of transcripts specific for the CXCR2 ELR-positive chemokine ligands CXCL1, CXCL2, and CXCL5 within the brain. Treatment of JHMV-infected mice with anti-CXCR2 blocking antibody reduced PMN trafficking into the CNS by >95%, dampened MMP-9 activity, and abrogated blood-brain-barrier (BBB) breakdown. Correspondingly, CXCR2 neutralization resulted in diminished infiltration of virus-specific T cells, an inability to control viral replication within the brain, and 100% mortality. Blocking CXCR2 signaling did not impair the generation of virus-specific T cells, indicating that CXCR2 is not required to tailor anti-JHMV T cell responses. Evaluation of mice in which CXCR2 is genetically silenced (CXCR2−/− mice) confirmed that PMNs neither expressed CXCR2 nor migrated in response to ligands CXCL1, CXCL2, or CXCL5 in an in vitro chemotaxis assay. Moreover, JHMV infection of CXCR2−/− mice resulted in an approximate 60% reduction of PMN migration into the CNS, yet these mice survived infection and controlled viral replication within the brain. Treatment of JHMV-infected CXCR2−/− mice with anti-CXCR2 antibody did not modulate PMN migration nor alter viral clearance or mortality, indicating the existence of compensatory mechanisms that facilitate sufficient migration of PMNs into the CNS in the absence of CXCR2. Collectively, these findings highlight a previously unappreciated role for ELR-positive chemokines in enhancing host defense during acute viral infections of the CNS.

CXCR2 Signaling Protects Oligodendrocytes and Restricts Demyelination in a Mouse Model of Viral-Induced Demyelination

Background The functional role of ELR-positive CXC chemokines during viral – induced demyelination was assessed. Inoculation of the neuroattenuated JHM strain of mouse hepatitis virus (JHMV) into the CNS of susceptible mice results in an acute encephalomyelitis that evolves into a chronic demyelinating disease, modeling white matter pathology observed in the human demyelinating disease Multiple Sclerosis. Methodology/Principal Findings JHMV infection induced the rapid and sustained expression of transcripts specific for the ELR (+) chemokine ligands CXCL1 and CXCL2, as well as their binding receptor CXCR2, which was enriched within the spinal cord during chronic infection. Inhibiting CXCR2 signaling with neutralizing antiserum significantly (p<0.03) delayed clinical recovery. Moreover, CXCR2 neutralization was associated with an increase in the severity of demyelination that was independent of viral recrudescence or modulation of neuroinflammation. Rather, blocking CXCR2 was associated with increased numbers of apoptotic cells primarily within white matter tracts, suggesting that oligodendrocytes were affected. JHMV infection of enriched oligodendrocyte progenitor cell (OPC) cultures revealed that apoptosis was associated with elevated expression of cleaved caspase 3 and muted Bcl-2 expression. Inclusion of CXCL1 within JHMV infected cultures restricted caspase 3 cleavage and increased Bcl-2 expression that was associated with a significant (p<0.001) decrease in apoptosis. CXCR2 deficient oligodendrocytes were refractory to CXCL1 mediated protection from JHMV – induced apoptosis, readily activating caspase 3 and down regulating Bcl-2. Conclusion/Significance These findings highlight a previously unappreciated role for CXCR2 signaling in protecting oligodendrocyte lineage cells from apoptosis during inflammatory demyelination initiated by viral infection of the CNS.

CD8+ Memory T Cells Appear Exhausted within Hours of Acute Virus Infection

CD8+ memory T cells are abundant and are activated in a near-synchronous manner by infection, thereby providing a unique opportunity to evaluate the coordinate functional and phenotypic changes that occur in vivo within hours of viral challenge. Using two disparate virus challenges of mice, we show that splenic CD8+ memory T cells rapidly produced IFN-γ in vivo; however, within 18–24 h, IFN-γ synthesis was terminated and remained undetectable for ≥48 h. A similar on/off response was observed in CD8+ memory T cells in the peritoneal cavity. Cessation of IFN-γ production in vivo occurred despite the continued presence of immunostimulatory viral Ag, indicating that the initial IFN-γ response had been actively downregulated and that the cells had been rendered refractory to subsequent in vivo Ag contact. Downregulation of IFN-γ synthesis was accompanied by the upregulation of inhibitory receptor expression on the T cells, and ex vivo analyses using synthetic peptides revealed a concurrent hierarchical loss of cytokine responsiveness (IL-2, then TNF, then IFN-γ) taking place during the first 24 h following Ag contact. Thus, within hours of virus challenge, CD8+ memory T cells display the standard hallmarks of T cell exhaustion, a phenotype that previously was associated only with chronic diseases and that is generally viewed as a gradually developing and pathological change in T cell function. Our data suggest that, instead, the “exhaustion” phenotype is a rapid and normal physiological T cell response.

The Biology of Persistent Infection: Inflammation and Demyelination following Murine Coronavirus Infection of the Central Nervous System.

Multiple Sclerosis (MS) is an immune-mediated demyelinating disease of humans. Although causes of MS are enigmatic, underlying elements contributing to disease development include both genetic and environmental factors. Recent epidemiological evidence has pointed to viral infection as a trigger to initiating white matter damage in humans. Mouse hepatitis virus (MHV) is a positive strand RNA virus that, following intracranial infection of susceptible mice, induces an acute encephalomyelitis that later resolves into a chronic fulminating demyelinating disease. Immune cell infiltration into the central nervous system is critical both to quell viral replication and instigate demyelination. Recent efforts by our laboratory and others have focused upon strategies capable of enhancing remyelination in response to viral-induced demyelination, both by dampening chronic inflammation and by surgical engraftment of remyelination - competent neural precursor cells.

Antigen-Specific Naive CD8+ T Cells Produce a Single Pulse of IFN-γ In Vivo within Hours of Infection, but without Antiviral Effect

In vitro studies have shown that naive CD8+ T cells are unable to express most of their effector proteins until after at least one round of cell division has taken place. We have reassessed this issue in vivo and find that naive CD8+ T cells mount Ag-specific responses within hours of infection, before proliferation has commenced. Newly activated naive Ag-specific CD8+ T cells produce a rapid pulse of IFN-γ in vivo and begin to accumulate granzyme B and perforin. Later, in vivo cytolytic activity is detectable, coincident with the initiation of cell division. Despite the rapid development of these functional attributes, no antiviral effect was observed early during infection, even when the cells are present in numbers similar to those of virus-specific memory cells. The evolutionary reason for the pulse of IFN-γ synthesis by naive T cells is uncertain, but the lack of antiviral impact suggests that it may be regulatory.

Wild-type coxsackievirus infection dramatically alters the abundance, heterogeneity, and immunostimulatory capacity of conventional dendritic cells in vivo.

In vitro studies have shown that enteroviruses employ strategies that may impair the ability of DCs to trigger T cell immunity, but it is unclear how these viruses affect DCs in vivo. Here, we evaluate the effects of wild-type (wt) coxsackievirus B3 on DCs in vitro and in a murine model in vivo. Although CVB3 does not productively infect the vast majority of DCs, virus infection profoundly reduces splenic conventional DC numbers and diminishes their capacity to prime naïve CD8(+) T cells in vitro. In contrast to recombinant CVB3, highly pathogenic wt virus infection significantly diminishes the hosts capacity to mount T cell responses, which is temporally associated with the loss of CD8α(+) DCs. Our findings demonstrate that enterovirus infection substantially alters the number, heterogeneity, and stimulatory capacity of DCs in vivo, and these dramatic immunomodulatory effects may weaken the hosts capacity to mount antiviral T cell responses.