T-lymphocytes are critical for protection against respiratory infections, such as
and influenza virus, with chemokine receptors playing an important role in directing these cells to the lungs. CXCR6 ...is expressed by activated T-lymphocytes and its ligand, CXCL16, is constitutively expressed by the bronchial epithelia, suggesting a role in T-lymphocyte recruitment and retention. However, it is unknown whether CXCR6 is required in responses to pulmonary infection, particularly on CD4
T-lymphocytes. Analysis of CXCR6-reporter mice revealed that in naïve mice, lung leukocyte expression of CXCR6 was largely restricted to a small population of T-lymphocytes, but this population was highly upregulated after either infection. Nevertheless, pulmonary infection of CXCR6-deficient mice with
or recombinant influenza A virus expressing P25 peptide (rIAV-P25), an I-A
-restricted epitope from the immunodominant mycobacterial antigen, Ag85B, demonstrated that the receptor was redundant for recruitment of T-lymphocytes to the lungs. Interestingly, CXCR6-deficiency resulted in reduced bacterial burden in the lungs 6 weeks after
infection, and reduced weight loss after rIAV-P25 infection compared to wild type controls. This was paradoxically associated with a decrease in Th1-cytokine responses in the lung parenchyma. Adoptive transfer of P25-specific CXCR6-deficient T-lymphocytes into WT mice revealed that this functional change in Th1-cytokine production was not due to a T-lymphocyte intrinsic mechanism. Moreover, there was no reduction in the number or function of CD4
and CD8
tissue resident memory cells in the lungs of CXCR6-deficient mice. Although CXCR6 was not required for T-lymphocyte recruitment or retention in the lungs, CXCR6 influenced the kinetics of the inflammatory response so that deficiency led to increased host control of
and influenza virus.
Oxidative stress caused by excessive reactive oxygen species production is implicated in influenza A virus-induced lung disease. Glutathione peroxidase (GPx)-1 is an antioxidant enzyme that may ...protect lungs from such damage. The objective of this study was to determine if GPx-1 protects the lung against influenza A virus-induced lung inflammation in vivo. Male wild-type (WT) or GPx-1(-/-) mice were inoculated with HKx31 (H3N2, 1 × 10(4) plaque-forming units), and bronchoalveolar lavage fluid (BALF)/lung compartments were analyzed on Days 3 and 7 after infection for inflammatory marker expression, histology, and viral titer. WT mice infected with HKx31 had significantly more BALF total cells, macrophages, neutrophils, and lymphocytes at Days 3 and 7 compared with naive WT animals (n = 5-8; P < 0.05). However, infected GPx-1(-/-) mice had significantly more BALF inflammation, which included more total cells, macrophages, and neutrophils, compared with WT mice, and this was abolished by treatment with the GPx mimetic ebselen. BALF inflammation persisted in GPx-1(-/-) mice on Day 10 after infection, and GPx-1(-/-) mice had significantly more influenza-specific CD8(+) T cells in spleen compared with WT mice (n = 3-4; P < 0.05). Infected GPx-1(-/-) mice had greater peribronchial and parenchymal inflammation than WT mice, and viral titer was significantly reduced in GPx-1(-/-) mice at Day 3 (n = 5; P < 0.05). Gene expression analysis revealed that infected GPx-1(-/-) mice had higher whole lung TNF-α, macrophage inflammatory protein (MIP)-1α, MIP-2, KC, and matrix metalloproteinase (MMP)-12 mRNA compared with infected WT mice. GPx-1(-/-) mice had more MIP-2 protein in BALF at Day 3 and more active MMP-9 protease in BALF at Days 3 and 7 than WT mice. These data indicate that GPx-1 reduces influenza A virus-induced lung inflammation.
Interferons (IFN) are pleiotropic cytokines essential for defense against infection, but the identity and tissue distribution of IFN-responsive cells in vivo are poorly defined. In this study, we ...generate a mouse strain capable of reporting IFN-signaling activated by all three types of IFNs and investigate the spatio-temporal dynamics and identity of IFN-responding cells following IFN injection and influenza virus infection. Despite ubiquitous expression of IFN receptors, cellular responses to IFNs are highly heterogenous in vivo and are determined by anatomical site, cell type, cellular preference to individual IFNs, and activation status. Unexpectedly, type I and II pneumocytes, the primary target of influenza infection, exhibit striking differences in the strength and temporal dynamics of IFN signaling associated with differential susceptibility to the viral infection. Our findings suggest that time- and cell-type-dependent integration of distinct IFN signals govern the specificity and magnitude of IFN responses in vivo.
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•M1Red mouse reports on cellular responses to all three types of interferons (IFNs)•Hematopoietic progenitors and myeloid cells highly respond to IFN stimulation•Respiratory influenza virus infection induces local and systemic IFN signaling•Influenza-susceptible pneumocytes respond poorly to IFNs early during infection
Stifter et al. generated an Irgm1 reporter mouse sensitive to induction by all three types of IFNs. They show that cellular responses to IFNs are highly heterogenous in vivo. Furthermore, different types of IFNs act in a cell-type-dependent manner to convey synergistic, antagonistic, or non-redundant signaling during influenza infection.
Cleavage of versican by ADAMTS5 removes the ECM blockade, allowing migration (top panel). ...versican accumulation in the absence of ADAMTS enzyme activity results in T cell clustering (bottom ...panel). Lung tissue and MLNs were removed from influenza virus infection C57.BL/6 and Vcan+/hdf mice and processed to generate single cell suspensions at day 10 p.i. for analysis of influenza-specific immunity. (E) Influenza-specific DbNP366-374+ CD8+ and DbPA224-233+ CD8+ tetramer positive T cells in pooled MLN were enumerated at day 10 p.i. (F) CD8+ T cell functionality was measured using ICS to assess influenza-specific DbNP366-374+IFNγ+CD8+ and DbPA224-233+IFNγ+CD8+ T cell responses at day 10 p.i. The results are expressed as means ± SD or as pooled means (MLN data) and statistical significance (relative to C57.BL/6 mice) determined by a Student’s t test (*p ≤ 0.05, ***p ≤ 0.005 relative to C57.BL/6, n = 5 representing three individual experiments).
The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in ...vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.
Reverse genetics systems allow artificial generation of non-segmented and segmented negative-sense RNA viruses, like influenza viruses, entirely from cloned cDNA. Since the introduction of reverse ...genetics systems over a decade ago, the ability to generate 'designer' influenza viruses in the laboratory has advanced both basic and applied research, providing a powerful tool to investigate and characterise host-pathogen interactions and advance the development of novel therapeutic strategies. The list of applications for reverse genetics has expanded vastly in recent years. In this review, we discuss the development and implications of this technique, including the recent controversy surrounding the generation of a transmissible H5N1 influenza virus. We will focus on research involving the identification of viral protein function, development of live-attenuated influenza virus vaccines, host-pathogen interactions, immunity and the generation of recombinant influenza virus vaccine vectors for the prevention and treatment of infectious diseases and cancer.
Influenza virus-specific CD8+ T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one ...influenza A virus (H1N1) can prime for a secondary CD8+ T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+ T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2Db MHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and $Ig^{-/-}\>(\mu MT)$ mice were more susceptible to challenge with the single NP HKx31 influenza A virus (HK)-NP and PA (HK-PA) mutants, but unlike the Ig-/- mice, Ig+/+ mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor $K^bPB1_{703}\>CD8^+$ T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+ T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.
The rapid recall of influenza virus-specific CD8⁺ T cell effector function is protective, although our understanding of T cell memory remains incomplete. Recent debate has focused particularly on the ...CD62L lymph node homing receptor. The present analysis shows that although functional memory can be established from both CD62Lhi and CD62Llo CD8⁺ T cell subsets soon after initial encounter between naïve precursors and antigen, the optimal precursors are CD8⁺CD44hiCD25lo immune lymphocytes isolated from draining lymph nodes on day 3.5 after influenza virus infection. Analysis of primed T cells at different times after challenge indicates that the capacity to transfer memory is diminished at the peak of the primary cytotoxic T lymphocyte response, challenging speculations that the transition to memory first requires full differentiation to effector status. It seems that location rather than CD62Lhi/lo phenotype may be the more profitable focus for further dissection of the early establishment of T cell memory.
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