Differential requirements by CD4+ and CD8+ T cells for soluble and membrane TNF in control of Francisella tularensis live vaccine strain intramacrophage growth

During primary infection with intracellular bacteria, the membrane-associated form of TNF provides some TNF functions, but the relative contributions during memory responses are not well-characterized. In this study, we determined the role of T cell-derived secreted and membrane-bound TNF (memTNF) during adaptive immunity to Francisella tularensis live vaccine strain (LVS). Although transgenic mice expressing only the memTNF were more susceptible to primary LVS infection than wild-type (WT) mice, LVS-immune WT and memTNF mice both survived maximal lethal secondary Francisella challenge. Generation of CD44(high) memory T cells and clearance of bacteria were similar, although more IFN-gamma and IL-12(p40) were produced by memTNF mice. To examine T cell function, we used an in vitro tissue coculture system that measures control of LVS intramacrophage growth by LVS-immune WT and memTNF-T cells. LVS-immune CD4(+) and CD8(+) T cells isolated from WT and memTNF mice exhibited comparable control of LVS growth in either normal or TNF-alpha knockout macrophages. Although the magnitude of CD4(+) T cell-induced macrophage NO production clearly depended on TNF, control of LVS growth by both CD4(+) and CD8(+) T cells did not correlate with levels of nitrite. Importantly, intramacrophage LVS growth control by CD8(+) T cells, but not CD4(+) T cells, was almost entirely dependent on T cell-expressed TNF, and required stimulation through macrophage TNFRs. Collectively, these data demonstrate that T cell-expressed memTNF is necessary and sufficient for memory T cell responses to this intracellular pathogen, and is particularly important for intramacrophage control of bacterial growth by CD8(+) T cells.     

Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.     

Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin

TNF and lymphotoxin-alpha (LT alpha) may act at various stages of the host response to Mycobacterium tuberculosis. To dissect the effects of TNF independent of LT alpha, we have used C57BL/6 mice with a disruption of the TNF gene alone (TNF-/-). Twenty-one days following aerosol M. tuberculosis infection there was a marked increase in the number of organisms in the lungs of TNF-/- mice, and by 28-35 days all animals had succumbed, with widespread dissemination of M. tuberculosis. In comparison with the localized granulomas containing activated macrophages and T cells in lungs and livers of C57BL/6 wild-type (wt) mice, cellular infiltrates in TNF-/- mice were poorly formed, with extensive regions of necrosis and neutrophilic infiltration of the alveoli. Phenotypic analysis of lung homogenates demonstrated similar numbers of CD4+ and CD8+ T cells in TNF-/- and wt mice, but in TNF-deficient mice the lymphocytes were restricted to perivascular and peribronchial areas rather than colocated with macrophages in granulomas. T cells from TNF-/- mice retained proliferative and cytokine responses to purified protein derivative, and delayed-type hypersensitivity to purified protein derivative was demonstrable. Macrophages within the lungs of TNF-/- and wt mice showed similar levels of MHC class II and inducible nitric oxide synthase expression, and levels of serum nitrite were comparable. Thus, the enhanced susceptibility of TNF-/- is not compensated for by the presence of LT alpha, and the critical role of TNF is not in the activation of T cells and macrophages but in the local organization of granulomas.     

Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV-/-, FucT-VII-/-, FucT-IV-/-/FucT-VII-/-, or wild-type control mice. In lymph nodes, infected FucT-IV-/-/FucT-VII-/- and, to a lesser extent, FucT-VII-/- mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV-/-/FucT-VII-/- mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.     

TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.     

The interaction between IL-18 and IL-18 receptor limits the magnitude of protective immunity and enhances pathogenic responses following infection with intracellular bacteria

The binding of IL-18 to IL-18Rα induces both proinflammatory and protective functions during infection, depending on the context in which it occurs. IL-18 is highly expressed in the liver of wild-type (WT) C57BL/6 mice following lethal infection with highly virulent Ixodes ovatus ehrlichia (IOE), an obligate intracellular bacterium that causes acute fatal toxic shock-like syndrome. In this study, we found that IOE infection of IL-18Rα(-/-) mice resulted in significantly less host cell apoptosis, decreased hepatic leukocyte recruitment, enhanced bacterial clearance, and prolonged survival compared with infected WT mice, suggesting a pathogenic role for IL-18/IL-18Rα in Ehrlichia-induced toxic shock. Although lack of IL-18R decreased the magnitude of IFN-γ producing type-1 immune response, enhanced resistance of IL-18Rα(-/-) mice against Ehrlichia correlated with increased proinflammatory cytokines at sites of infection, decreased systemic IL-10 production, increased frequency of protective NKT cells producing TNF-α and IFN-γ, and decreased frequency of pathogenic TNF-α-producing CD8(+) T cells. Adoptive transfer of immune WT CD8(+) T cells increased bacterial burden in IL-18Rα(-/-) mice following IOE infection. Furthermore, rIL-18 treatment of WT mice infected with mildly virulent Ehrlichia muris impaired bacterial clearance and enhanced liver injury. Finally, lack of IL-18R signal reduced dendritic cell maturation and their TNF-α production, suggesting that IL-18 might promote the adaptive pathogenic immune responses against Ehrlichia by influencing T cell priming functions of dendritic cells. Together, these results suggested that the presence or absence of IL-18R signals governs the pathogenic versus protective immunity in a model of Ehrlichia-induced immunopathology.     

Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guérin infection in the absence of secreted TNF and lymphotoxin-alpha

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-alpha (LT-alpha) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-alpha(-/-), TNF(-/-), and LT-alpha(-/-) mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-alpha(-/-) mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-alpha and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.     

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40(-/-) mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.     

B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis

Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell(-/-)) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell(-/-) mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell(-/-) mice show increased production of IL-10 in the lungs, whereas IFN-gamma, TNF-alpha, and IL-10R remain unchanged from wild type. B cell(-/-) mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell(-/-) mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.     

Attenuated EAN in TNF-α deficient mice is associated with an altered balance of M1/M2 macrophages

The role of tumor necrosis factor (TNF)-α and its receptors in neuroautoimmune and neuroinflammatory diseases has been controversial. On the basis of our previous studies, we hereby aimed to further clarify TNF-α's mechanism of action and to explore the potential role of TNF-α receptor (TNFR)1 as a therapeutic target in experimental autoimmune neuritis (EAN). EAN was induced by immunization with P0 peptide 180-199 in TNF-α knockout (KO) mice and anti-TNFR1 antibodies were used to treat EAN. Particularly, the effects of TNF-α deficiency and TNFR1 blockade on macrophage functions were investigated. The onset of EAN in TNF-α KO mice was markedly later than that in wild type (WT) mice. From day 14 post immunization, the clinical signs of TNF-α KO mice were significantly milder than those of their WT counterparts. Further, we showed that the clinical severity of WT mice treated with anti-TNFR1 antibodies was less severe than that of the control WT mice receiving PBS. Nevertheless, no difference with regard to the clinical signs of EAN or inflammatory infiltration in cauda equina was seen between TNF-α KO and WT mice with EAN after blockade of TNFR1. Although TNF-α deficiency did not alter the proliferation of lymphocytes in response to either antigenic or mitogenic stimuli, it down-regulated the production of interleukin (IL)-12 and nitric oxide (NO), and enhanced the production of IL-10 in macrophages. Increased ratio of regulatory T cells (Tregs) and reduced production of interferon (IFN)-γ in cauda equina infiltrating cells, and elevated levels of IgG2b antibodies against P0 peptide 180-199 in sera were found in TNF-α KO mice with EAN. In conclusion, TNF-α deficiency attenuates EAN via altering the M1/M2 balance of macrophages.     

CD137 differentially regulates innate and adaptive immunity against Mycobacterium tuberculosis

Protective immunity against Mycobacterium tuberculosis is primarily mediated by the interaction of antigen-specific T cells and antigen presenting cells, which often depends on the interplay of cytokines produced by these cells. Costimulatory signals represent a complex network of receptor-ligand interactions that qualitatively and quantitatively influence immune responses. Thus, here we investigated the function of CD137 and CD137L, molecules known to have a central role in immune regulation, during human tuberculosis (TB). We demonstrated that M. tuberculosis antigen stimulation increased both CD137 and CD137L expression on monocytes and NK cells from TB patients and healthy donors, but only up-regulated CD137 on T lymphocytes. Blockage of the CD137 pathway enhanced the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α produced by monocytes and NK against M. tuberculosis. In contrast, CD137 blockage significantly decreased the specific degranulation of CD8(+) T cells and the percentage of specific IFN-γ and TNF-α producing lymphocytes against the pathogen. Furthermore, inhibition of the CD137 pathway markedly increased T-cell apoptosis. Taken together, our results demonstrate that CD137:CD137L interactions regulate the innate and adaptive immune response of the host against M. tuberculosis.     

Fc gamma receptors regulate immune activation and susceptibility during Mycobacterium tuberculosis infection

The critical role of cellular immunity during tuberculosis (TB) has been extensively studied, but the impact of Abs upon this infection remains poorly defined. Previously, we demonstrated that B cells are required for optimal protection in Mycobacterium tuberculosis-infected mice. FcgammaR modulate immunity by engaging Igs produced by B cells. We report that C57BL/6 mice deficient in inhibitory FcgammaRIIB (RIIB-/-) manifested enhanced mycobacterial containment and diminished immunopathology compared with wild-type controls. These findings corresponded with enhanced pulmonary Th1 responses, evidenced by increased IFN-gamma-producing CD4+ T cells, and elevated expression of MHC class II and costimulatory molecules B7-1 and B7-2 in the lungs. Upon M. tuberculosis infection and immune complex engagement, RIIB-/- macrophages produced more of the p40 component of the Th1-promoting cytokine IL-12. These data strongly suggest that FcgammaRIIB engagement can dampen the TB Th1 response by attenuating IL-12p40 production or activation of APCs. Conversely, C57BL/6 mice lacking the gamma-chain shared by activating FcgammaR had enhanced susceptibility and exacerbated immunopathology upon M. tuberculosis challenge, associated with increased production of the immunosuppressive cytokine IL-10. Thus, engagement of distinct FcgammaR can divergently affect cytokine production and susceptibility during M. tuberculosis infection.     

Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-alpha (LTalpha), is still unknown. Upon oral infection with T. gondii, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and-early after infection-splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice as compared with WT animals. Frequencies of parasite-specific IFN-gamma-producing T cells, intracerebral and splenic IFN-gamma production, and T. gondii-specific IgM and IgG titers in LTalpha(-/-) and TNF/LTalpha(-/-) mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-), but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-gamma-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTalpha for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTalpha and TNF for control of intracerebral toxoplasms.     

Nitric oxide synthase-2 modulates chemokine production by Trypanosoma cruzi-infected cardiac myocytes

An intense inflammatory process is associated with Trypanosoma cruzi infection. We investigated the mediators that trigger leukocyte activation and migration to the heart of infected mice. It is known that nitric oxide (NO) modulates the inflammatory response. During T. cruzi infection, increased concentrations of NO are produced by cardiac myocytes (CMs) in response to IFN-gamma and TNF. Here, we investigated whether NO, IFN-gamma and TNF regulate chemokine production by T. cruzi-infected CMs. In addition, we examined the effects of the NOS2 deficiency on chemokine expression both in cultured CMs and in hearts obtained from infected mice. After infection of cultured WT CMs with T. cruzi, the addition of IFN-gamma and TNF increased both mRNA and protein levels of the chemokines CXCL1, CXCL2, CCL2, CCL3, CCL4 and CCL5. Interestingly, T. cruzi-infected NOS2-deficient CMs produced significantly higher levels of CCL2, CCL4, CCL5 and CXL2 in the presence of IFN-gamma and TNF. Infection of NOS2-null mice resulted in a significant increase in the expression of both chemokine mRNA and protein levels in the heart of, compared with hearts obtained from, infected WT mice. Our data indicate that NOS2 is a potent modulator of chemokine expression which is critical to triggering the generation of the inflammatory infiltrate in the heart during T. cruzi infection.     

Tumor necrosis factor-α receptor type 1, not type 2, mediates its acute responses in the kidney

Acute administration of tumor necrosis factor-α (TNF-α) resulted in decreases in renal blood flow (RBF) and glomerular filtration rate (GFR) but induced diuretic and natriuretic responses in mice. To define the receptor subtypes involved in these renal responses, experiments were conducted to assess the responses to human recombinant TNF-α (0.3 ng·min(-1)·g body wt(-1) iv infusion for 75 min) in gene knockout (KO) mice for TNF-α receptor type 1 (TNFαR1 KO, n = 5) or type 2 (TNFαR2 KO, n = 6), and the results were compared with those obtained in corresponding wild-type [WT (C57BL/6), n = 6] mice. Basal levels of RBF (PAH clearance) and GFR (inulin clearance) were similar in TNFαR1 KO, but were lower in TNFαR2 KO, than WT mice. TNF-α infusion in WT mice decreased RBF and GFR but caused a natriuretic response, as reported previously. In TNFαR1 KO mice, TNF-α infusion failed to cause such vasoconstrictor or natriuretic responses; rather, there was an increase in RBF and a decrease in renal vascular resistance. Similar responses were also observed with infusion of murine recombinant TNF-α in TNFαR1 KO mice (n = 5). However, TNF-α infusion in TNFαR2 KO mice caused changes in renal parameters qualitatively similar to those observed in WT mice. Immunohistochemical analysis in kidney slices from WT mice demonstrated that while both receptor types were generally located in the renal vascular and tubular cells, only TNFαR1 was located in vascular smooth muscle cells. There was an increase in TNFαR1 immunoreactivity in TNFαR2 KO mice, and vice versa, compared with WT mice. Collectively, these functional and immunohistological findings in the present study demonstrate that the activation of TNFαR1, not TNFαR2, is mainly involved in mediating the acute renal vasoconstrictor and natriuretic actions of TNF-α.     

CXCL9 and CXCL10 expression are critical for control of genital herpes simplex virus type 2 infection through mobilization of HSV-specific CTL and NK cells to the nervous system

CXCL9 and CXCL10 mediate the recruitment of T lymphocytes and NK cells known to be important in viral surveillance. The relevance of CXCL10 in comparison to CXCL9 in response to genital HSV-2 infection was determined using mice deficient in CXCL9 (CXCL9-/-) and deficient in CXCL10 (CXCL10-/-) along with wild-type (WT) C57BL/6 mice. An increased sensitivity to infection was found in CXCL10-/- mice in comparison to CXCL9-/- or WT mice as determined by detection of HSV-2 in the CNS at day 3 postinfection. However, by day 7 postinfection both CXCL9-/- and CXCL10-/- mice possessed significantly higher viral titers in the CNS in comparison to WT mice consistent with mortality (18-35%) of these mice within the first 7 days after infection. Even though CXCL9-/- and CXCL10-/- mice expressed elevated levels of CCL2, CCL3, CCL5, and CXCL1 in the spinal cord in comparison to WT mice, there was a reduction in NK cell and virus-specific CD8+ T cell mobilization to this tissue, suggesting CXCL9 and CXCL10 are critical for recruitment of these effector cells to the spinal cord following genital HSV-2 infection. Moreover, leukocytes from the spinal cord but not from draining lymph nodes or spleens of infected CXCL9-/- or CXCL10-/- mice displayed reduced CTL activity in comparison to effector cells from WT mice. Thus, the absence of CXCL9 or CXCL10 expression significantly alters the ability of the host to control genital HSV-2 infection through the mobilization of effector cells to sites of infection.     

Dominant-negative inhibitors of soluble TNF attenuate experimental arthritis without suppressing innate immunity to infection

TNF is a pleiotropic cytokine required for normal development and function of the immune system; however, TNF overexpression also induces inflammation and is associated with autoimmune diseases. TNF exists as both a soluble and a transmembrane protein. Genetic studies in mice have suggested that inflammation in disease models involves soluble TNF (solTNF) and that maintenance of innate immune function involves transmembrane TNF (tmTNF). These findings imply that selective pharmacologic inhibition of solTNF may be anti-inflammatory and yet preserve innate immunity to infection. To address this hypothesis, we now describe dominant-negative inhibitors of TNF (DN-TNFs) as a new class of biologics that selectively inhibits solTNF. DN-TNFs blocked solTNF activity in human and mouse cells, a human blood cytokine release assay, and two mouse arthritis models. In contrast, DN-TNFs neither inhibited the activity of human or mouse tmTNF nor suppressed innate immunity to Listeria infection in mice. These results establish DN-TNFs as the first selective inhibitors of solTNF, demonstrate that inflammation in mouse arthritis models is primarily driven by solTNF, and suggest that the maintenance of tmTNF activity may improve the therapeutic index of future anti-inflammatory agents.     

CCR5-deficient mice control Mycobacterium tuberculosis infection despite increased pulmonary lymphocytic infiltration

The control of Mycobacterium tuberculosis infection is dependent on the development of an adaptive immune response, which is mediated by granulomas. The granuloma is a dynamic structure that forms in the lung and consists primarily of macrophages and lymphocytes. For this structure to be effective in containment of the bacillus, it must develop in an organized and timely manner. The formation of the granuloma is dependent on recruitment of activated cells through adhesion molecules and chemokines. M. tuberculosis infection causes an increase in the expression of beta-chemokines CCL3, CCL4, and CCL5, and their receptor CCR5, in the lungs. In this study, we demonstrate that CCR5-transgenic knockout mice were capable of recruiting immune cells to the lung to form granulomas. CCR5(-/-) mice successfully induced a Th1 response and controlled infection. Unexpectedly, M. tuberculosis infection in these mice resulted in greater numbers of lymphocytes migrating to the lung and higher levels of many inflammatory cytokines, compared with wild-type mice, without apparent long-term detrimental effects. In the absence of CCR5, there were more dendritic cells in the lung-draining lymph nodes and more primed T lymphocytes in these mice. Bacterial numbers in the lymph nodes were also higher in CCR5(-/-) mice. Therefore, CCR5 may play a role in the migration of dendritic cells to and from the lymph nodes during M. tuberculosis infection.     

Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.