James A. Harker

Late Interleukin-6 Escalates T Follicular Helper Cell Responses and Controls a Chronic Viral Infection

A persisting wave of the cytokine interleukin-6 allows control of chronic viral infections. Multiple inhibitory molecules create a profoundly immunuosuppressive environment during chronic viral infections in humans and mice. Therefore, eliciting effective immunity in this context represents a challenge. Here, we report that during a murine chronic viral infection, interleukin-6 (IL-6) was produced by irradiation-resistant cells in a biphasic manner, with late IL-6 being absolutely essential for viral control. The underlying mechanism involved IL-6 signaling on virus-specific CD4 T cells that caused up-regulation of the transcription factor Bcl6 and enhanced T follicular helper cell responses at late, but not early, stages of chronic viral infection. This resulted in escalation of germinal center reactions and improved antibody responses. Our results uncover an antiviral strategy that helps to safely resolve a persistent infection in vivo.

Organ‐specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C. rodentium in situ and in real time. The bioluminescent C. rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo. We have demonstrated that primary colonization of the mouse by C. rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch. Following colonization of the caecum C. rodentium established a colonic infection. Clearance of C. rodentium ICC180 parallels the colonization dynamics, i.e. the caecum was first to be cleared followed by the colon. A bioluminescent eae (encoding the outer membrane adhesin intimin) C. rodentium mutant failed to establish long‐term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.

Alveolar Macrophages Are a Major Determinant of Early Responses to Viral Lung Infection but Do Not Influence Subsequent Disease Development

ABSTRACT Macrophages are abundant in the lower respiratory tract. They play a central role in the innate response to infection but may also modulate excessive inflammation. Both macrophages and ciliated epithelial cells respond to infection by releasing soluble mediators, leading to the recruitment of innate and adaptive effector cells. To study the role of lung macrophages in acute respiratory viral infection, we depleted them by the inhalation of clodronate liposomes in an established mouse model of respiratory syncytial virus (RSV) disease. Infection caused an immediate local release of inflammatory cytokines and chemokines, peaking on day 1, which was virtually abolished by clodronate liposome treatment. Macrophage depletion inhibited the activation (days 1 to 2) and recruitment (day 4) of natural killer (NK) cells and enhanced peak viral load in the lung (day 4). However, macrophage depletion did not affect the recruitment of activated CD4 or CD8 T cells, weight loss, or virus-induced changes in lung function. Therefore, lung macrophages play a central role in the early responses to viral infection but have remarkably little effect on the adaptive response occurring at the time of peak disease severity.

CD25+ Natural Regulatory T Cells Are Critical in Limiting Innate and Adaptive Immunity and Resolving Disease following Respiratory Syncytial Virus Infection

ABSTRACT Regulatory CD4+ T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4+ Foxp3+ CD25+ natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25+ natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-γ). CD25+ natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-γ and tumor necrosis factor alpha (TNF-α) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.

The role of T cells in the enhancement of respiratory syncytial virus infection severity during adult reinfection of neonatally sensitized mice.

ABSTRACT Respiratory syncytial virus (RSV) is the major cause of infantile bronchiolitis and hospitalization. Severe RSV disease is associated with the development of wheezing in later life. In a mouse model of the delayed effects of RSV, the age at primary infection determines responses to reinfection in adulthood. During primary RSV infection, neonatal BALB/c mice developed only mild disease and recruited CD8 cells that were defective in gamma interferon production. Secondary reinfection of neonatally primed mice caused enhanced inflammation and profuse lung T-cell recruitment. CD4 cell depletion during secondary RSV challenge attenuated disease (measured by weight loss); depletion of CD8 cells also markedly attenuated disease severity but enhanced lung eosinophilia, and depletion of both CD4 and CD8 cells together completely abrogated weight loss. Depletion of CD8 (but not CD4) cells during primary neonatal infection was protective against weight loss during adult challenge. Therefore, T cells, in particular CD8 T cells, play a central role in the outcome of neonatal infection by enhancing disease during secondary challenge. These findings demonstrate a crucial role for T cells in the regulation of immune responses after neonatal infection.

RSV-Induced Bronchial Epithelial Cell PD-L1 Expression Inhibits CD8+ T Cell Nonspecific Antiviral Activity

Respiratory syncytial virus (RSV) is a major cause of bronchiolitis in infants. It is also responsible for high morbidity and mortality in the elderly. Programmed death ligands (PD-Ls) on antigen-presenting cells interact with receptors on T cells to regulate immune responses. The programmed death receptor-ligand 1/programmed death receptor 1 (PD-L1-PD-1) pathway is inhibitory in chronic viral infections, but its role in acute viral infections is unclear. We hypothesized that bronchial epithelial cell (BEC) expression of PD-Ls would inhibit local effector CD8(+) T cell function. We report that RSV infection of primary human BECs strongly induces PD-L1 expression. In a co-culture system of BECs with purified CD8(+) T cells, we demonstrated that RSV-infected BECs increased CD8(+) T cell activation, proliferation, and antiviral function. Blocking PD-L1 on RSV-infected BECs co-cultured with CD8(+) T cells enhanced CD8(+) T cell IFN-γ, IL-2, and granzyme B production. It also decreased the virus load of the BECs. Based on our findings, we believe therapeutic strategies that target the PD-L1-PD-1 pathway might increase antiviral immune responses to RSV and other acute virus infections.

Cell-Intrinsic IL-27 and gp130 Cytokine Receptor Signaling Regulates Virus-Specific CD4+ T Cell Responses and Viral Control during Chronic Infection

The outcome of chronic viral infections, which affect millions of people worldwide, is greatly dependent on CD4⁺ T cells. Here we showed that T cell-specific ablation of the common interleukin-6 (IL-6) family receptor, gp130, profoundly compromised virus-specific CD4⁺ T cell survival, T follicular helper responses, and IL-21 production at late stages of chronic lymphocytic choriomeningitis virus (LCMV) infection. These effects were cell intrinsic for CD4⁺ T cells and were accompanied by a reduction of CD8⁺ T cells, antibodies, and a severe failure in viral control. We identified IL-27 as a gp130 cytokine that promoted antiviral CD4⁺ T cell accumulation in vivo and that rapidly induced IL-21 ex vivo. Furthermore, IL-27R was critical for control of persistent LCMV in vivo. These results reveal that gp130 cytokines (particularly IL-27) are key regulators of CD4⁺ T cell responses during an established chronic viral infection, empowering both humoral and cytotoxic immunity.

Innate and Adaptive Immune Regulation During Chronic Viral Infections

Chronic viral infections represent a unique challenge to the infected host. Persistently replicating viruses outcompete or subvert the initial antiviral response, allowing the establishment of chronic infections that result in continuous stimulation of both the innate and adaptive immune compartments. This causes a profound reprogramming of the host immune system, including attenuation and persistent low levels of type I interferons, progressive loss (or exhaustion) of CD8(+) T cell functions, and specialization of CD4(+) T cells to produce interleukin-21 and promote antibody-mediated immunity and immune regulation. Epigenetic, transcriptional, posttranscriptional, and metabolic changes underlie this adaptation or recalibration of immune cells to the emerging new environment in order to strike an often imperfect balance between the host and the infectious pathogen. In this review we discuss the common immunological hallmarks observed across a range of different persistently replicating viruses and host species, the underlying molecular mechanisms, and the biological and clinical implications.

Interleukin 18 coexpression during respiratory syncytial virus infection results in enhanced disease mediated by natural killer cells.

ABSTRACT Respiratory syncytial virus (RSV) causes bronchiolitis, the main cause of infantile hospitalization. Immunity against reinfection is poor, and there is great interest in boosting vaccine responses using live vectors expressing host cytokines. We therefore constructed a recombinant RSV expressing murine interleukin 18 (RSV/IL-18), a cytokine capable of inducing strong antiviral immune responses. In vitro RSV/IL-18 replicated at wild-type levels and produced soluble IL-18. In naïve BALB/c mice, RSV/IL-18 infection significantly increased both IL-18 mRNA and protein and attenuated the peak viral load 3-fold. Despite a reduced viral load, RSV/IL-18 infection caused a biphasic weight loss at days 2 and 6 postinfection that was not seen in wild-type infection. Day 2 disease was associated with enhanced pulmonary natural killer (NK) cell numbers and activity and was prevented by NK cell depletion during infection; day 6 disease was correlated with CD8 T-cell recruitment and was enhanced by NK cell depletion. IL-18 expression during priming also enhanced RSV-specific antibody responses and T-cell responses on secondary RSV infection. Therefore, while IL-18 boosted antiviral immunity and reduced the viral load, its coexpression worsened disease. This is the first recombinant RSV with this property, and these are the first studies to demonstrate that NK cells can induce pathology during pulmonary viral infections.

Genetic Susceptibility to the Delayed Sequelae of Neonatal Respiratory Syncytial Virus Infection Is MHC Dependent

Respiratory syncytial virus (RSV) is a major cause of respiratory morbidity, resulting in hospitalization for bronchiolitis in some infected infants that is associated with wheeze in later life. Genetic factors are known to affect the severity of the sequelae after RSV infection, but the complexity of the temporal and genetic effects makes it difficult to analyze this response in studies in man. Therefore, we developed a murine genetic model to analyze the sequelae occurring after RSV infection in early life. Haplotype-based genetic analysis of interstrain differences in severity identified the MHC as an important genetic determinant. This was confirmed by analysis of responses in congenic mice with different MHC haplotypes. We also found that susceptible strains had high CD8 levels during secondary infection. Analysis of first filial generation, second filial generation, and back-cross progeny produced by intercrossing resistant (H-2k, C3H/HeN) and sensitive (H-2b, BALB/c) strains indicated that susceptibility to sequelae after RSV infection was dominantly inherited but also segregated in a non-MHC–dependent manner. Thus, MHC haplotype and its effect on CD8 cell response is an important determinant of the outcome of neonatal RSV infection.