Lawrence W. Kummer

Systemic but Not Local Infections Elicit Immunosuppressive IL-10 Production by Natural Killer Cells

Surviving infection represents a balance between the proinflammatory responses needed to eliminate the pathogen, and anti-inflammatory signals limiting damage to the host. IL-10 is a potent immunosuppressive cytokine whose impact is determined by the timing and localization of release. We show that NK cells rapidly express IL-10 during acute infection with diverse rapidly disseminating pathogens. The proinflammatory cytokine IL-12 was necessary and sufficient for NK cell induction of IL-10. NK cells from mice with systemic parasitic infection inhibited dendritic cell release of IL-12 in an IL-10-dependent manner, and NK cell depletion resulted in elevated serum IL-12. These data suggest an innate, negative feedback loop in which IL-12 limits its own production by eliciting IL-10 from NK cells. In contrast to disseminating pathogens, locally restricted infections did not elicit NK cell IL-10. Thus systemic infections uniquely engage NK cells in an IL-10-mediated immunoregulatory circuit that functions to alleviate inflammation.

Cell-Mediated Protection against Pulmonary Yersinia pestis Infection

ABSTRACT Pulmonary infection with the bacterium Yersinia pestis causes pneumonic plague, an often-fatal disease for which no vaccine is presently available. Antibody-mediated humoral immunity can protect mice against pulmonary Y. pestis infection, an experimental model of pneumonic plague. Little is known about the protective efficacy of cellular immunity. We investigated the cellular immune response to Y. pestis in B-cell-deficient μMT mice, which lack the capacity to generate antibody responses. To effectively prime pulmonary cellular immunity, we intranasally vaccinated μMT mice with live replicating Y. pestis. Vaccination dramatically increased survival of μMT mice challenged intranasally with a lethal Y. pestis dose and significantly reduced bacterial growth in pulmonary, splenic, and hepatic tissues. Vaccination also increased numbers of pulmonary T cells, and administration of T-cell-depleting monoclonal antibodies at the time of challenge abrogated vaccine-induced survival. Moreover, the transfer of Y. pestis-primed T cells to naive μMT mice protected against lethal intranasal challenge. These findings establish that vaccine-primed cellular immunity can protect against pulmonary Y. pestis infection and suggest that vaccines promoting both humoral and cellular immunity will most effectively combat pneumonic plague.

Gamma Interferon, Tumor Necrosis Factor Alpha, and Nitric Oxide Synthase 2, Key Elements of Cellular Immunity, Perform Critical Protective Functions during Humoral Defense against Lethal Pulmonary Yersinia pestis Infection

ABSTRACT Pulmonary infection by Yersinia pestis causes pneumonic plague, a rapidly progressing and often fatal disease. To aid the development of safe and effective pneumonic plague vaccines, we are deciphering mechanisms used by the immune system to protect against lethal pulmonary Y. pestis infection. In murine pneumonic plague models, passive transfer of convalescent-phase sera confers protection, as does active vaccination with live Y. pestis. Here, we demonstrate that protection by either protocol relies upon both gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) cytokines classically associated with type 1 cellular immunity. In both protocols, abrogating IFN-γ or TNF-α activity significantly decreases survival and increases the bacterial burden in pulmonary, splenic, and hepatic tissues. Neutralization of either cytokine also counteracts challenge-induced, vaccination-dependent upregulation of nitric oxide synthase 2 (NOS2). Moreover, genetic depletion of NOS2 suppresses protection conferred by serotherapy. We conclude that IFN-γ, TNF-α, and NOS2, key elements of cellular immunity, perform critical protective functions during humoral defense against lethal pulmonary Y. pestis challenge. These observations strongly suggest that plague vaccines should strive to maximally prime both cellular and humoral immunity.

Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection

Natural regulatory T cells (Tregs) constitutively express the IL-2R α-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4+ T cell IFN-γ production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4+ T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings.

Protective Roles for Fibrin, Tissue Factor, Plasminogen Activator Inhibitor-1, and Thrombin Activatable Fibrinolysis Inhibitor, but Not Factor XI, during Defense against the Gram-Negative Bacterium Yersinia enterocolitica

Septic infections dysregulate hemostatic pathways, prompting coagulopathy. Nevertheless, anticoagulant therapies typically fail to protect humans from septic pathology. The data reported in this work may help to explain this discrepancy by demonstrating critical protective roles for coagulation leading to fibrin deposition during host defense against the Gram-negative bacterium Yersinia enterocolitica. After i.p. inoculation with Y. enterocolitica, fibrinogen-deficient mice display impaired cytokine and chemokine production in the peritoneal cavity and suppressed neutrophil recruitment. Moreover, both gene-targeted fibrinogen-deficient mice and wild-type mice treated with the anticoagulant coumadin display increased hepatic bacterial burden and mortality following either i.p. or i.v. inoculation with Y. enterocolitica. Mice with low tissue factor activity succumb to yersiniosis with a phenotype similar to fibrin(ogen)-deficient mice, whereas factor XI-deficient mice show wild-type levels of resistance. Mice deficient in plasminogen activator inhibitor-1 or thrombin-activatable fibrinolysis inhibitor display modest phenotypes, but mice deficient in both plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor succumb to yersiniosis with a phenotype resembling fibrin(ogen)-deficient mice. These findings demonstrate critical protective roles for the tissue factor-dependent extrinsic coagulation pathway during host defense against bacteria and caution that therapeutics targeting major thrombin-generating or antifibrinolytic pathways may disrupt fibrin-mediated host defense during Gram-negative sepsis.

IL-17 contributes to cell-mediated defense against pulmonary Yersinia pestis infection.

Pneumonic plague is one of the world’s most deadly infectious diseases. The causative bacterium, Yersinia pestis, has the potential to be exploited as a biological weapon, and no vaccine is available. Vaccinating B cell-deficient mice with D27-pLpxL, a live attenuated Y. pestis strain, induces cell-mediated protection against lethal pulmonary Y. pestis challenge. In this article, we demonstrate that prime/boost vaccination with D27-pLpxL confers better protection than prime-only vaccination. The improved survival does not result from enhanced bacterial clearance but is associated with increased levels of IL-17 mRNA and protein in the lungs of challenged mice. The boost also increases pulmonary numbers of IL-17–producing CD4 T cells. Interestingly, most of these cells simultaneously produce canonical type 1 and type 17 cytokines; most produce IL-17 and TNF-α, and many produce IL-17, TNF-α, and IFN-γ. Neutralizing IL-17 counteracts the improved survival associated with prime/boost vaccination without significantly impacting bacterial burden. Thus, IL-17 appears to mediate the enhanced protection conferred by booster immunization. Although neutralizing IL-17 significantly reduces neutrophil recruitment to the lungs of mice challenged with Y. pestis, this impact is equally evident in mice that receive one or two immunizations with D27-pLpxL, suggesting it cannot suffice to account for the improved survival that results from booster immunization. We conclude that IL-17 plays a yet to be identified role in host defense that enhances protection against pulmonary Y. pestis challenge, and we suggest that pneumonic plague vaccines should aim to induce mixed type 1 and type 17 cellular responses.

Live attenuated influenza vaccine (LAIV) impacts innate and adaptive immune responses

Influenza A infection induces a massive inflammatory response in the lungs that leads to significant illness and increases the susceptibility to secondary bacterial pneumonia. The most efficient way to prevent influenza infection is through vaccination. While inactivated vaccines induce protective levels of serum antibodies to influenza hemaglutinin (HA) and neuraminidase (NA) surface proteins, these are strain specific and offer little protection against heterosubtypic influenza viruses. In contrast, live attenuated influenza vaccines (LAIVs) induce a T cell response in addition to antibody responses against HA and NA surface proteins. Importantly, LAIV vaccination induces a response in a mouse model that protects against illness due to heterosubtypic influenza strains. While it is not completely clear what is the mechanism of action of LAIV heterosubtypic protection in humans, it has been shown that LAIV induces heterosubtypic protection in mice that is dependent upon a Type 1 immune response and requires CD8 T cells. In this study, we show that LAIV-induced immunity leads to significantly reduced viral titers and inflammatory responses in the lungs of mice following heterosubtypic infection. Not only are viral titers reduced in LAIV vaccinated mice, the amounts of inflammatory cytokines and chemokines in lung tissue are significantly lower. Additionally, we show that LAIV vaccination of healthy adults also induces a robust Type 1 memory response including the production of chemokines and cytokines involved in T cell activation and recruitment. Thus, our results indicate that LAIV vaccination functions by inducing immune memory which can act to modulate the immune response to subsequent heterosubtypic challenge by influencing both innate and adaptive responses.

Infection-Stimulated Fibrin Deposition Controls Hemorrhage and Limits Hepatic Bacterial Growth during Listeriosis

ABSTRACT Bacterial infections are major causes of human mortality. The activation of coagulation pathways leading to the deposition of insoluble fibrin frequently accompanies bacterial infection, and much attention has focused upon the pathological attributes of infection-stimulated fibrin deposition. Nevertheless, here we present conclusive evidence that infection-stimulated fibrin deposition can perform critical protective functions during bacterial infection. Specifically, we demonstrate that coagulation-impaired fibrin(ogen)-deficient mice, in comparison with genetically matched control mice, display increased mortality upon peritoneal infection with the gram-positive facultative intracellular bacterium Listeria monocytogenes. To distinguish effects of fibrinogen from those of fibrin, we treat wild-type mice with warfarin, an anticoagulant that suppresses fibrin formation without impacting fibrinogen levels. Warfarin treatment exacerbates listeriosis, suggesting that fibrin is the key mediator of protection. With regard to the underlying protective mechanisms, we demonstrate that fibrin(ogen) suppresses anemia, reduces hemorrhagic pathology, and limits bacterial growth during listeriosis. Despite confirming a prior report that fibrin(ogen) promotes the peritoneal clearance of the extracellular bacterium Staphylococcal aureus, we demonstrate that fibrin(ogen) plays little role in controlling peritoneal numbers of L. monocytogenes bacteria or the dissemination of L. monocytogenes bacteria from the peritoneal cavity. Rather, fibrin(ogen) primarily limits the growth of these intracellular bacteria within hepatic tissue. While the pathological potential of excessive infection-stimulated fibrin deposition is well appreciated, our findings reveal that fibrin can function protectively, via multiple mechanisms, during bacterial infection.

Antibodies and cytokines independently protect against pneumonic plague

Yersinia pestis causes pneumonic plague, an exceptionally virulent disease for which we lack a safe and effective vaccine. Antibodies specific for the Y. pestis F1 and LcrV proteins can protect mice against pulmonary Y. pestis infection. We demonstrate that neutralizing tumor necrosis factor-alpha (TNFalpha) and gamma-interferon (IFNgamma) abrogates this protection at sub-optimal levels of F1- or LcrV-specific antibody, but not at optimal levels. Moreover, we demonstrate that endogenous TNFalpha and IFNgamma confer measurable protection in the complete absence of protective antibodies. These findings indicate that antibodies and cytokines independently protect against pneumonic plague and suggest that surrogate assays for plague vaccine efficacy should consider both the level of vaccine-induced antibody and the capacity of vaccine recipients to produce TNFalpha and IFNgamma upon exposure to Y. pestis.

D27-pLpxL, an Avirulent Strain of Yersinia pestis, Primes T Cells That Protect against Pneumonic Plague

ABSTRACT Vaccinating with live, conditionally attenuated, pigmentation (Pgm)-deficient Yersinia pestis primes T cells that protect mice against pneumonic plague. However, Pgm-deficient strains are not considered safe for human use because they retain substantial virulence in animal models. Y. pestis strains engineered to express Escherichia coli LpxL are avirulent owing to constitutive production of lipopolysaccharide with increased Toll-like receptor 4-activating ability. We generated an LpxL-expressing Pgm-deficient strain (D27-pLpxL) and demonstrate here that this avirulent strain retains the capacity to prime protective T cells. Compared with unvaccinated controls, mice immunized intranasally with live D27-pLpxL exhibit a decreased bacterial burden and increased survival when challenged intranasally with virulent Y. pestis. T cells provide a substantial degree of this protection, as vaccine efficacy is maintained in B-cell-deficient μMT mice unless those animals are depleted of CD4 and CD8 T cells at the time of challenge. Upon challenge with Y. pestis, pulmonary T-cell numbers decline in naive mice, whereas immunized mice show increased numbers of CD44high CD43high effector T cells and T cells primed to produce tumor necrosis factor alpha and gamma interferon; neutralizing these cytokines at the time of challenge abrogates protection. Immunization does not prevent dissemination of Y. pestis from the lung but limits bacterial growth and pathology in visceral tissue, apparently by facilitating formation of granuloma-like structures. This study describes a new model for studying T-cell-mediated protection against pneumonic plague and demonstrates the capacity for live, highly attenuated, Y. pestis vaccine strains to prime protective memory T-cell responses safely.