Expression of suppressor of cytokine signaling 1 (SOCS1) impairs viral clearance and exacerbates lung injury during influenza infection.

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1-/- mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1-/-IFN-γ-/- mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1-/-IFN-γ-/- mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ-/- or WT animals. Enhanced viral clearance in SOCS1-/-IFN-γ-/- mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1-/-IFN-γ-/- mice had no significant effect on their enhanced resistance to influenza infection, while CD8+ splenocytes from SOCS1-/-IFN-γ-/- mice were sufficient to rescue RAG1-/- animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1-/- mice reconstituted with SOCS1-/-IFN-γ-/- adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury

Nox2-derived oxidative stress results in inefficacy of antibiotics against post-influenza S. aureus pneumonia.

Clinical post-influenza Staphylococcus aureus pneumonia is characterized by extensive lung inflammation associated with severe morbidity and mortality even after appropriate antibiotic treatment. In this study, we show that antibiotics rescue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2)-deficient mice but fail to fully protect WT animals from influenza and S. aureus coinfection. Further experiments indicate that the inefficacy of antibiotics against coinfection is attributable to oxidative stress-associated inflammatory lung injury. However, Nox2-induced lung damage during coinfection was not associated with aggravated inflammatory cytokine response or cell infiltration but rather caused by reduced survival of myeloid cells. Specifically, oxidative stress increased necrotic death of inflammatory cells, thereby resulting in lethal damage to surrounding tissue. Collectively, our results demonstrate that influenza infection disrupts the delicate balance between Nox2-dependent antibacterial immunity and inflammation. This disruption leads to not only increased susceptibility to S. aureus infection, but also extensive lung damage. Importantly, we show that combination treatment of antibiotic and NADPH oxidase inhibitor significantly improved animal survival from coinfection. These findings suggest that treatment strategies that target both bacteria and oxidative stress will significantly benefit patients with influenza-complicated S. aureus pneumonia

A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes.

UNLABELLED: To enhance the research capabilities of investigators interested in Staphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen. IMPORTANCE: Infections caused by Staphylococcus aureus cause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) strain repository. In addition, complementary resources, including a website (http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigating S. aureus pathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems

Expression of Suppressor of Cytokine Signaling 1 (SOCS1) Impairs Viral Clearance and Exacerbates Lung Injury during Influenza Infection

<div><p>Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1^−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1^−/−IFN-γ^−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1^−/−IFN-γ^−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ^−/− or WT animals. Enhanced viral clearance in SOCS1^−/−IFN-γ^−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1^−/−IFN-γ^−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8⁺ splenocytes from SOCS1^−/−IFN-γ^−/− mice were sufficient to rescue RAG1^−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1^−/− mice reconstituted with SOCS1^−/−IFN-γ^−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.</p></div

CD8⁺ T cells from SOCS1^−/−IFN-γ^−/− mice protect against influenza infection.

<p>(<b>A</b>) Survival, (<b>B</b>) viral burdens, and (<b>C</b>) albumin levels (5–7 mice/group) at 11 dpi in C57BL/6 RAG1^−/− mice after i.n. infection of 50 PFU PR8 influenza virus. Mice were i.p injected with 10⁷ CD8⁺ T cells isolated from WT, IFN-γ^−/− or SOCS1^−/−IFN-γ^−/− mice 10 days before infection. In (B&C), <i>P</i><0.01, ANOVA; *<i>P</i><0.05, **, <i>P</i><0.01, Tukey's multiple comparisons test. Data in (A&B) were combined from two independent experiments. Data in (C) are representative of two experiments.</p

Influenza infection improves adaptive immune responses in SOCS1^−/−IFN-γ^−/− mice.

<p>(<b>A</b>) Numbers of airway CD4⁺ T cells, (<b>B</b>) CD8⁺ T cells, and (<b>C</b>) BALF H1N1-specific IgM and IgG levels in C57BL/6 WT, IFN-γ^−/− and SOCS1^−/−IFN-γ^−/− mice after 50 PFU PR8 infection (4 mice/group). In (A&B), <i>P</i><0.001, ANOVA; *<i>P</i><0.05, **, <i>P</i><0.01, ***, <i>P</i><0.001, Tukey's multiple comparisons test. The data for each time point were repeated in at least two independent experiments.</p

Decreased accumulation of inflammatory cells in SOCS1^−/−IFN-γ^−/− mice at the resolution phase of influenza infection.

<p>(<b>A</b>) Numbers of CD11b⁺, CD11b⁺Ly6B⁻ and CD11b⁺Ly6B⁺ myeloid cell subsets, (<b>B</b>) flow cytometry analysis of airway CD11b⁺, CD11b⁺Ly6B⁻ and CD11b⁺Ly6B⁺ myeloid cell subsets, and (<b>C</b>) numbers of CD11b⁺Ly6B⁺Ly6G⁺ neutrophils and CD11b⁺Ly6B⁺Ly6G⁻Ly6C⁺ inflammatory monocytes at 11 dpi in C57BL/6 WT, IFN-γ^−/− and SOCS1^−/−IFN-γ^−/− mice after 50 PFU PR8 infection (4 mice/group). In (A), <i>P</i><0.001, ANOVA; <i>P</i><0.05, and **, <i>P</i><0.01, and ***, <i>P</i><0.001, Tukey's multiple comparisons test, the data for each time point were repeated in at least two independent experiments. Data in (B&C) are representative of at least two experiments.</p

Influenza infection enhances DC recruitment in SOCS1^−/−IFN-γ^−/− mice.

<p>(<b>A</b>) Numbers of BALF cells (4 mice/group) and (<b>B</b>) flow cytometry analysis of airway CD11c⁺ cell subsets at 7 dpi in C57BL/6 WT, IFN-γ^−/− and SOCS1^−/−IFN-γ^−/− mice after 50 PFU PR8 infection (3–4 mice/group). In (A) <i>P</i><0.001, ANOVA; *<i>P</i><0.05, ***, <i>P</i><0.001 relative to WT and IFN-γ^−/− mice, Tukey's multiple comparisons test, the data for each time point were repeated in at least two independent experiments. Data in (B) are representative of two independent experiments.</p

Antiviral immune responses in RAG1^−/− mice reconstituted with SOCS1^−/−IFN-γ^−/− adaptive immune cells.

<p>(<b>A</b>) Airway viral burdens (5–6 mice/group), (<b>B</b>) T cell numbers at 7 dpi (4–5 mice/group), (<b>C</b>) H1N1-specific antibody responses (4–6 mice/group), (<b>D</b>) cytokine responses (5–6 mice/group) at 7 dpi, and (<b>E</b>) albumin levels (5–6 mice/group) at 11 dpi in C57BL/6 RAG1^−/− mice after i.n. infection of 50 PFU PR8 influenza virus. Mice were i.p injected with 2×10⁷ IFN-γ^−/− or SOCS1^−/−IFN-γ^−/− splenocytes 10 weeks before influenza infection. In (A), <i>P</i><0.001, ANOVA; ***, <i>P</i><0.001, Tukey's multiple comparisons test. In (B), ***, <i>P</i><0.001, <i>t</i> test. In (C), <i>P</i><0.01, ANOVA; *<i>P</i><0.05, **, <i>P</i><0.01, Tukey's multiple comparisons test. The data for each time point were repeated in at least two independent experiments.</p