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The coronavirus disease 2019 (COVID-19) pandemic is a rapidly evolving global emergency that continues to strain healthcare systems. Emerging research describes a plethora of patient factors-including demographic, clinical, immunologic, hematological, biochemical, and radiographic findings-that may be of utility to clinicians to predict COVID-19 severity and mortality. We present a synthesis of the current literature pertaining to factors predictive of COVID-19 clinical course and outcomes. Findings associated with increased disease severity and/or mortality include age > 55 years, multiple pre-existing comorbidities, hypoxia, specific computed tomography findings indicative of extensive lung involvement, diverse laboratory test abnormalities, and biomarkers of end-organ dysfunction. Hypothesis-driven research is critical to identify the key evidence-based prognostic factors that will inform the design of intervention studies to improve the outcomes of patients with COVID-19 and to appropriately allocate scarce resources., (© 2020 John Wiley & Sons, Ltd.)

Antiviral defense in the liver during acute infection with the hepatotropic virus murine cytomegalovirus (MCMV) involves complex cytokine and cellular interactions. However, the mechanism of viral sensing in the liver that promotes these cytokine and cellular responses has remained unclear. Studies here were undertaken to investigate the role of nucleic acid-sensing Toll-like receptors (TLRs) in initiating antiviral immunity in the liver during infection with MCMV. We examined the host response of UNC93B1 mutant mice, which do not signal properly through TLR3, TLR7 and TLR9, to acute MCMV infection to determine whether liver antiviral defense depends on signaling through these molecules. Infection of UNC93B1 mutant mice revealed reduced production of systemic and liver proinflammatory cytokines including IFN-α, IFN-γ, IL-12 and TNF-α when compared to wild-type. UNC93B1 deficiency also contributed to a transient hepatitis later in acute infection, evidenced by augmented liver pathology and elevated systemic alanine aminotransferase levels. Moreover, viral clearance was impaired in UNC93B1 mutant mice, despite intact virus-specific CD8+ T cell responses in the liver. Altogether, these results suggest a combined role for nucleic acid-sensing TLRs in promoting early liver antiviral defense during MCMV infection.

Various cell types in both lymphoid and non-lymphoid tissues produce the anti-inflammatory cytokine interleukin (IL)-10 during murine cytomegalovirus (MCMV) infection. The functions of IL-10 in the liver during acute infection and the cells that generate this cytokine at this site have not been extensively investigated. In this study, we demonstrate that the production of IL-10 in the liver is elevated in C57BL/6 mice during late acute MCMV infection. Using IL-10 green fluorescence protein (GFP) reporter knock-in mice, designated IL-10-internal ribosomal entry site (IRES)-GFP-enhanced reporter (tiger), NK cells are identified as major IL-10 expressing cells in the liver after infection, along with T cells and other leukocytes. In the absence of IL-10, mice exhibit marked elevations in proinflammatory cytokines and in the numbers of mononuclear cells and lymphocytes infiltrating the liver during this infection. IL-10-deficiency also enhances liver injury without improving viral clearance from this site. Collectively, the results indicate that IL-10-producing cells in the liver provide protection from collateral injury by modulating the inflammatory response associated with MCMV infection.

Monocytes/macrophages are critical early innate immune responders during murine CMV (MCMV) infection. It has been established that inflammatory monocyte/macrophages are released from the bone marrow and into the peripheral blood before entry into infected tissue sites. We previously reported a role for IFN-alpha/beta in promotion of CCR2-mediated recruitment of monocyte/macrophages into the liver in response to MCMV infection. However, the mechanisms that support the migration of monocyte/macrophages from the bone marrow and into the peripheral blood under conditions of MCMV infection have not been elucidated. Herein, we demonstrate an accumulation of monocyte/macrophages in the bone marrow of MCMV-infected CCR2-deficient mice, whereas circulating monocyte/macrophages are profoundly diminished. The CCR2 ligands MCP-1, MCP-3, and MCP-5 are detected in bone marrow and in serum from MCMV-infected mice. Furthermore, bone marrow leukocytes from naive mice produce high levels of MCP-1 and MCP-5, and moderate levels of MCP-3, when stimulated with recombinant IFN-alpha in culture. We identify bone marrow F4/80(+) cells as major producers of MCP-1, MCP-3, and MCP-5. Moreover, induction of CCR2 ligands is dependent on IFN-alpha/beta-mediated signals and MCMV infection. Taken together, the results reveal a critical role for inflammatory cytokines in stimulating production of CCR2-binding chemokines from F4/80(+) cells in the bone marrow, and they suggest that local production of chemokines supports monocyte/macrophage egress from the bone marrow into the blood during a virus infection.

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-alphabeta. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-alphabeta in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-alpha in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-alpha production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-alpha, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-alpha production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-alpha in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-gamma, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88(-/-) mice. Additionally, MyD88(-/-) but not TLR9(-/-) mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.

Although studies blocking the Fas pathway indicate it can decrease organ damage while improving septic (cecal ligation and puncture, CLP) mouse survival, little is known about how Fas-Fas ligand (FasL) interactions mediate this protection at the tissue level. Here, we report that although Fas expression on splenocytes and hepatocytes is up-regulated by CLP and is inhibited by in vivo short interfering RNA, FasL as well as the frequency of CD8(+) T cells are differentially altered by sepsis in the spleen (no change in FasL, decreased percentage of CD8(+) and CD4(+) T cells) versus the liver (increased FasL expression on CD8(+) T cells and increase in percentage/number). Adoptive transfer of CLP FasL(+/+) versus FasL(-/-) mouse liver CD8(+) T cells to severe combined immunodeficient or RAG1(-/-) recipient mice indicated that these cells could induce inflammation. The FasL-mediated cytotoxic capacity of these septic mouse liver CD8(+) T cells was shown by their ability to damage directly cultured hepatocytes. Finally, although CD8(-/-) mice exhibited a reduction in both CLP-induced liver active caspase-3 staining and blood interleukin-6 levels, only FasL(-/-) (but not CD8(-/-)) protected the septic mouse spleen from increasing apoptosis. Thus, although truncating Fas-FasL signaling ameliorates many untoward effects of sepsis, the pathological mode of action is distinct at the tissue level.

Innate inflammatory events promoting antiviral defense in the liver against murine cytomegalovirus (MCMV) infection have been characterized. However, the mechanisms that regulate the selective recruitment of inflammatory T lymphocytes to the liver during MCMV infection have not been defined. The studies presented here demonstrate the expression of monokine induced by gamma interferon (IFN-gamma; Mig/CXCL9) and IFN-gamma-inducible protein 10 (IP-10/CXCL10) in liver leukocytes and correlate their production with the infiltration of MCMV-specific CD8 T cells into the liver. Antibody-mediated neutralization of CXCL9 and CXCL10 and studies using mice deficient in CXCR3, the primary known receptor for these chemokines, revealed that CXCR3-dependent mechanisms promote the infiltration of virus-specific CD8 T cells into the liver during acute infection with MCMV. Furthermore, CXCR3 functions augmented the hepatic accumulation of CD8 T-cell IFN-gamma responses to MCMV. Evaluation of protective functions demonstrated enhanced pathology that overlapped with transient increases in virus titers in CXCR3-deficient mice. However, ultimate viral clearance and survival were not compromised. Thus, CXCR3-mediated signals support the accumulation of MCMV-specific CD8 T cells that contribute to, but are not exclusively required for, protective responses in a virus-infected tissue site.

Leukocyte trafficking to the central nervous system (CNS), regulated in part by chemokines, determines severity of the demyelinating diseases multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). To examine chemokine receptor CX3CR1 in EAE, we studied CX3CR1(GFP/GFP) mice, in which CX3CR1 targeting by insertion of Green Fluorescent Protein (GFP) allowed tracking of CX3CR1+ cells in CX3CR1(+/GFP) animals and cells destined to express CX3CR1 in CX3CR1(GFP/GFP) knockouts. NK cells were markedly reduced in the inflamed CNS of CX3CR1-deficient mice with EAE, whereas recruitment of T cells, NKT cells and monocyte/macrophages to the CNS during EAE did not require CX3CR1. Impaired recruitment of NK cells in CX3CR1(GFP/GFP) mice was associated with increased EAE-related mortality, nonremitting spastic paraplegia and hemorrhagic inflammatory lesions. The absence of CD1d did not affect the severity of EAE in CX3CR1(GFP/GFP) mice, arguing against a role for NKT cells. Accumulation of NK cells in livers of wild-type (WT) and CX3CR1(GFP/GFP) mice with cytomegalovirus hepatitis was equivalent, indicating that CX3CL1 mediated chemoattraction of NK cells was relatively specific for the CNS. These results are the first to define a chemokine that governs NK cell migration to the CNS, and the findings suggest novel therapeutic manipulation of CX3CR1+ NK cells.

The complex interplays between cytokines and chemokines are emerging as key communication signals in the shaping of innate and adaptive immune responses against foreign pathogens, including viruses. In particular, the virus-induced expression of cytokine and chemokine profiles drives the recruitment and activation of immune effector cells to sites of tissue infection. Under the conditions of infection with murine cytomegalovirus (MCMV), a herpesvirus with pathogenic potential, early immune functions are essential in the control of virus replication and virus-induced pathology. The coordinated MCMV-induced cytokine and chemokine responses promote effective natural killer (NK) cell recruitment and function, and ultimately MCMV clearance. The studies highlighted in this chapter illustrate in vivo pathways mediated by innate cytokines in regulating chemokine responses that are vital for localized antiviral defenses.

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.

Dendritic cells (DCs) present microbial antigens to T cells and provide inflammatory signals that modulate T cell differentiation. While the role of DCs in adaptive immunity is well established, their involvement in innate immune defenses is less well defined. We have identified a TNF/iNOS-producing (Tip)-DC subset in spleens of Listeria monocytogenes-infected mice that is absent from CCR2-deficient mice. The absence of Tip-DCs results in profound TNF and iNOS deficiencies and an inability to clear primary bacterial infection. CD8 and CD4 T cell responses to L. monocytogenes antigens are preserved in CCR2-deficient mice, indicating that Tip-DCs are not essential for T cell priming. Tip-DCs, as the predominant source of TNF and iNOS during L. monocytogenes infection, orchestrate and mediate innate immune defense against this intracellular bacterial pathogen.

Differentiation of dendritic cells (DCs) into particular subsets may act to shape innate and adaptive immune responses, but little is known about how this occurs during infections. Plasmacytoid dendritic cells (PDCs) are major producers of interferon (IFN)-alpha/beta in response to many viruses. Here, the functions of these and other splenic DC subsets are further analyzed after in vivo infection with murine cytomegalovirus (MCMV). Viral challenge induced PDC maturation, their production of high levels of innate cytokines, and their ability to activate natural killer (NK) cells. The conditions also licensed PDCs to efficiently activate CD8 T cells in vitro. Non-plasmacytoid DCs induced T lymphocyte activation in vitro. As MCMV preferentially infected CD8alpha+ DCs, however, restricted access to antigens may limit plasmacytoid and CD11b+ DC contribution to CD8 T cell activation. IFN-alpha/beta regulated multiple DC responses, limiting viral replication in all DC and IL-12 production especially in the CD11b+ subset but promoting PDC accumulation and CD8alpha+ DC maturation. Thus, during defense against a viral infection, PDCs appear specialized for initiation of innate, and as a result of their production of IFN-alpha/beta, regulate other DCs for induction of adaptive immunity. Therefore, they may orchestrate the DC subsets to shape endogenous immune responses to viruses.

The recruitment of immune effector cells to localized sites of infection is crucial for the effective delivery of innate immune mechanisms. Under the conditions of infections with murine cytomegalovirus (MCMV), a herpesvirus with pathogenic potential, early immune functions are essential in the control of virus replication and virus-induced pathology. Our studies have demonstrated that the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) is critical for natural killer (NK) cell inflammation and delivery of interferon (IFN)-gamma to mediate downstream protective responses against MCMV infection in liver. Moreover, IFN-alpha/beta-dependent mechanisms promote MIP-1alpha production and subsequently the accumulation of NK cells in liver. Taken together, the studies highlighted in this review define a unique in vivo pathway mediated by innate cytokines in regulating chemokine responses that are essential in the promotion of NK cell inflammation for localized antiviral defense. In addition, the downstream consequences of these events in enhancing endogenous adaptive immune responses will also be discussed. Overall, the innate cytokine/chemokine networks that are described emphasize the emerging importance of chemokine functions for protective immune responses during infection with viruses.

NK cell cytotoxicity, IFN-gamma expression, proliferation, and accumulation are rapidly induced after murine CMV infections. Under these conditions, the responses were shown to be elicited in overlapping populations. Nevertheless, there were distinct signaling molecule requirements for induction of functions within the subsets. IL-12/STAT4 was critical for NK cell IFN-gamma expression, whereas IFN-alphabeta/STAT1 were required for induction of cytotoxicity. The accumulation/survival of proliferating NK cells was STAT4-independent but required IFN-alphabeta/STAT1 induction of IL-15. Taken together, the results define the coordinated interactions between the cytokines IFN-alphabeta, IL-12, and IL-15 for activation of protective NK cell responses during viral infections, and emphasize these factors' nonredundant functions under in vivo physiological conditions.

Macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) is critical for liver NK cell inflammation and delivery of IFN-gamma to mediate downstream protective responses against murine cytomegalovirus (MCMV) infections. This system was used to evaluate the upstream contribution of the type 1 IFNs, IFN-alpha/beta, in promotion of MIP-1alpha production. Mice deficient in IFN-alpha/beta functions, as a result of mutation in the receptor for these cytokines (IFN-alpha/betaR(-)), were profoundly deficient in MIP-1alpha expression and accumulation of NK cells and macrophages in the liver and had increased sensitivity to MCMV infection. The cytokines themselves were responsible for the immunoregulatory effects, since administration of recombinant IFN-alpha (rIFN-alpha) to immunocompetent mice also induced these changes. IFN-alpha/beta was required for NK cell accumulation during infection, and MIP-1alpha was required for NK cell accumulation in response to administered rIFN-alpha. In vivo trafficking assays demonstrated a requirement for IFN-alpha/betaR signaling for leukocyte localization in, and delivery of MIP-1alpha-producing macrophages to, the liver. These results extend characterization of the cytokine and chemokine cascade required for protection against viral infections in tissues by defining IFN-alpha/beta-dependent mechanisms promoting MIP-1alpha production and the resulting hepatic accumulation of NK cells.

Viruses and viral components can be potent inducers of alpha/beta interferons (IFN-alpha/beta). In culture, IFN-alpha/beta prime for their own expression, in response to viruses, through interferon regulatory factor 7 (IRF-7) induction. The studies presented here evaluated the requirements for functional IFN receptors and the IFN signaling molecule STAT1 in IFN-alpha/beta induction during infections of mice with lymphocytic choriomeningitis virus (LCMV). At 24 h after infection, levels of induced IFN-alpha/beta in serum were reduced 90 to 95% in IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) and STAT1(-/-) mice compared to those in wild-type mice. However, at 48 h, these mice showed elevated expression in the serum whereas IFN-alpha/beta levels were still reduced >75% in IFN-alpha/betagammaR(-/-) mice even though the viral burden was heavy. Levels of IFN-beta, IFN-alpha4, and non-IFN-alpha4 subtype mRNA expression correlated with IFN-alpha/beta bioactivity, and all IFN-alpha/beta subtypes were coincidentally detectable. IRF-7 mRNA was induced under conditions of IFN-alpha/beta production, including late production in IFN-alpha/betaR(-/-) mice. These data demonstrate that the presence of the virus alone is not sufficient to induce IFN-alpha/beta during LCMV infection in vivo. Instead, autocrine amplification through the IFN-alpha/betaR is necessary for optimal induction. In the absence of a functional IFN-alpha/betaR, however, alternative mechanisms, independent of STAT1 but requiring a functional IFN-gammaR, take over.

Interferon (IFN)-alpha/beta and interleukin (IL)-12 are cytokines critical in defense against viruses, but their cellular sources and mechanisms of regulation for in vivo expression remain poorly characterized. The studies presented here identified a novel subset of dendritic cells (DCs) as major producers of the cytokines during murine cytomegalovirus (MCMV) but not lymphocytic choriomeningitis virus (LCMV) infections. These DCs differed from those activated by Toxoplasma antigen but were related to plasmacytoid cells, as assessed by their CD8alpha(+)Ly6G/C(+)CD11b(-) phenotype. Another DC subset (CD8alpha(2)Ly6G/C(-)CD11b(+)) also contributed to IL-12 production in MCMV-infected immunocompetent mice, modestly. However, it dramatically increased IL-12 expression in the absence of IFN-alpha/beta functions. Conversely, IFN-alpha/beta production was greatly reduced under these conditions. Thus, a cross-regulation of DC subset cytokine responses was defined, whereby secretion of type I IFNs by CD8alpha(+) DCs resulted in responses limiting IL-12 expression by CD11b(+) DCs but enhancing overall IFN-alpha/beta production. Taken together, these data indicate that CD8alpha(+)Ly6G/C(+)CD11b(-) DCs play important roles in limiting viral replication and regulating immune responses, through cytokine production, in some but not all viral infections. They also illustrate the plasticity of cellular sources for innate cytokines in vivo and provide new insights into the roles of IFNs in shaping immune responses to viruses.

NK cell functions for regulation of T cell responses were evaluated during acute viral infections. In vivo depletion studies established that the presence of NK cells in murine cytomegalovirus (MCMV)-infected immunocompetent mice negatively affected CD4 and CD8 T cell IFN-gamma expression, bromodeoxyuridine (BrdU) incorporation, and expansion. To evaluate NK cell effects, under conditions when NK cells do not control viral replication, experiments were performed using lymphocytic choriomeningitis virus (LCMV). Depletion of NK cells did not affect LCMV-elicited T cell responses in immunocompetent mice; however, the presence of NK cells did inhibit CD4 T cell IFN-gamma production, BrdU incorporation, and expansion in infected MHC class I- and CD8 T cell-deficient beta2M-/- mice. Together, the results reveal a previously unappreciated immunoregulatory role of NK cells for downstream T cell responses.

Macrophage inflammatory protein 1alpha (MIP-1alpha) promotes natural killer (NK) cell inflammation in livers during murine cytomegalovirus (MCMV) infections, and NK cell-produced interferon gamma (IFN-gamma) contributes to defense against MCMV infections. A specific role for local NK cell IFN-gamma production, however, has not been established. The importance of MIP-1alpha and NK cell-produced IFN-gamma in shaping endogenous immune responses and defense in different compartments was examined. MIP-1alpha deficiency profoundly decreased resistance to MCMV and was associated with dramatically reduced NK cell accumulation and IFN-gamma production in liver. MIP-1alpha-independent IFN-gamma responses were observed in serum and spleen, and infection-induced elevations in blood NK cell populations occurred in absence of the factor, but peak liver expression of another chemokine, the monokine induced by IFN-gamma (Mig), depended upon presence of MIP-1alpha, NK cells, and IFN-gamma. The Mig response was also important for viral resistance. Thus, serum cytokine responses are insufficient; MIP-1alpha is critical for NK cell migration and IFN-gamma delivery to mediate protection; and Mig induction in tissues is a downstream protective response resulting from the process. These results define a critical chemokine-to-cytokine-to-chemokine cascade required for defense during a viral infection establishing itself in tissues.

Natural killer (NK) cells are populations of lymphocytes that can be activated to mediate significant levels of cytotoxic activity and produce high levels of certain cytokines and chemokines. NK cells respond to and are important in defense against a number of different infectious agents. The first indications for this function came from the observations that virus-induced interferons alpha/beta (IFN-alpha and -beta) are potent inducers of NK cell-mediated cytotoxicity, and that NK cells are important contributors to innate defense against viral infections. In addition to IFN-alpha/beta, a wide range of other innate cytokines can mediate biological functions regulating the NK cell responses of cytotoxicity, proliferation, and gamma interferon (IFN-gamma) production. Certain, but not all, viral infections induce interleukin 12 (IL-12) to elicit NK cell IFN-gamma production and antiviral mechanisms. However, high levels of IFN-alpha/beta appear to be unique and/or uniquely dominant in the context of viral infections and act to regulate other innate responses, including induction of NK cell proliferation in vivo and overall negative regulation of IL-12 production. A detailed picture is developing of particular innate cytokines activating NK cell responses and their consorted effects in providing unique endogenous milieus promoting downstream adaptive responses, most beneficial in defense against viral infections.

Natural killer (NK) cells mediate defense against early murine cytomegalovirus (MCMV) infections in liver. The chemokine, macrophage inflammatory protein 1alpha (MIP-1alpha), can promote inflammatory responses. Our studies evaluated contributions of NK cells to early MCMV-induced liver inflammation and MIP-1alpha requirements for inflammation and delivery of antiviral defenses. NK cells were shown to be responsible for focal inflammation, and to be induced to migrate at high levels, in MCMV-infected livers. MIP-1alpha gene expression was elevated at coinciding times, and mice deficient in MIP-1alpha function were dramatically inhibited in both inflammatory and protective liver responses. The results precisely define MIP-1alpha-dependent steps required to achieve NK cell inflammation during, and mechanisms promoting defense against, viral infections in tissues.

The contribution of endogenous NK cells and cytokines to virus-induced liver pathology was evaluated during murine cytomegalovirus infections of mice. In immunocompetent C57BL/6 mice, the virus induced a self-limited liver disease characterized by hepatitis, with focal inflammation, and large grossly visible subcapsular necrotic foci. The inflammatory foci were most numerous and contained the greatest number of cells 3 days after infection; they colocalized with areas of viral antigen expression. The largest number of necrotic foci was found 2 days after infection. Overall hepatic damage, assessed as increased expression of liver enzymes in serum, accompanied the development of inflammatory and necrotic foci. Experiments with neutralizing antibodies demonstrated that although virus-induced tumor necrosis factor (TNF) can have antiviral effects, it also mediated significant liver pathology. TNF was required for development of hepatic necrotic foci and increased levels of liver enzymes in serum but not for increased numbers of inflammatory foci. The necrotic foci and liver enzyme indications of pathology occurred independently of NK and T cells, because mice rendered NK-cell deficient by treatment with antibodies, T- and B-cell-deficient Rag-/- mice, and NK- and T-cell-deficient E26 mice all manifested both parameters of disease. Development of necrotic foci and maximally increased levels of liver enzymes in serum also were TNF dependent in NK-cell-deficient mice. Moreover, in the immunodeficient E26 mice, virus-induced liver disease was progressive, with eventual death of the host, and neutralization of TNF significantly increased longevity. These results establish conditions separating hepatitis from significant liver damage and demonstrate a cytokine-mediated component to viral pathogenesis.

Studies were undertaken to characterize mechanisms for NK cell cytokine delivery in vivo. Conditions of systemic IFN-beta expression elicited by polyinosinic-polycytidylic acid (poly(I:C)) treatment or IFN-alpha beta production during lymphocytic choriomeningitis virus or murine cytomegalovirus infections resulted in profound splenic histologic changes, with relocalization of nucleated cells from red to white pulp regions. Cell-trafficking experiments, with fluorescently labeled populations, showed that poly(I:C) induced T/B cell-dependent leukocyte migration into white pulp regions. Splenic leukocytes prepared from severe combined immunodeficient (SCID) and bone marrow cells prepared from SCID or normal C57BL/6 mice revealed a unique poly(I:C)-induced accumulation of non-T/non-B cells along splenic red and white pulp region borders characteristic of marginal zones. Lymphocytic choriomeningitis virus and murine cytomegalovirus infections also induced this trafficking pattern. Ab treatments of normal and SCID cell donor mice to eliminate specific cell subsets demonstrated that the unique migration of non-T/non-B cells was dependent upon cells with the NK phenotype, NK1.1+ AGM1+F4/80-. In situ hybridization showed that, at early times after infections, small proportions of cells along red and white pulp divisions expressed high levels of IFN-gamma mRNA, and that these cells were depleted by protocols eliminating NK cells. The results suggest an exciting model for induction of NK cell trafficking from bone marrow to secondary compartments. This migratory pattern may localize NK cells to receive additional activation signals and/or to precisely deliver NK cell-produced cytokines.

Interleukin 12 (IL-12) doses in excess of 100 ng/d have been shown to induce profound immunotoxicities in mice infected with lymphocytic choriomeningitis virus (LCMV). These immunotoxicities are characterized by almost complete inhibition of virus-induced CD8+ T cell expansion and CTL activation, and up to 2 log increases in viral replication. They are accompanied by induction of serum tumor necrosis factor (TNF). The studies presented here were undertaken to characterize mechanisms for the IL-12-induced toxicities and to examine expression and function of TNF in this context. Several physiological changes were induced in IL-12-treated uninfected and dramatically elevated in IL-12-treated virus-infected mice. IL-12 induced (a) decreases in body weights, > 10% in uninfected and > 20% in LCMV-infected mice; (b) elevation of circulating glucocorticoid levels to > 10 micrograms/dl in uninfected and > 20 micrograms/dl in infected mice; and (c) decreases in thymic mass, > 30% in uninfected and up to 95% in infected mice. These changes are known to be associated with circulating TNF. Northern blot and in situ hybridization analyses demonstrated that IL-12 induced TNF-alpha expression and that LCMV infection synergized with IL-12 for induction of this factor. Antibodies neutralizing TNF reversed all of the IL-12-induced toxicities in LCMV-infected mice including the immunotoxicities against CD8+ T cells and anti-viral defenses. The TNF-mediated immunotoxicities appeared to result from an induced cellular sensitivity to the factor, as splenic leukocytes and CD8+ T cell subsets isolated from LCMV-infected mice were more sensitive to TNF-mediated cytotoxicity in culture than were equivalent populations prepared from uninfected mice. Experiments with the glucocorticoid type II receptor antagonist, RU486, demonstrated that endogenous glucocorticoids were secondary intermediaries in IL-12-induced thymic atrophy. Studies in IL-2-deficient mice showed that the synergism was dependent upon endogenous IL-2. The results delineate a unique mechanism of TNF-mediated toxicity. In addition, they have significant implications concerning potential detrimental consequences of in vivo TNF induction and of IL-12 administration for protective anti-viral responses.