Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 × 10⁷ PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2⁺, CD3⁺, CD4⁺, and CD8⁺ T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-γ)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-γ-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans, including possible detrimental secondary effects.     

Dexamethasone and indomethacin modify endotoxin-induced respiratory failure in pigs

We studied the porcine pulmonary response to endotoxemia before and after administration of nonsteroidal antiinflammatory drugs (NSAID, i.e., indomethacin or flunixin meglumine) or dexamethasone (DEX). Escherichia coli endotoxin was infused intravenously into anesthetized 10- to 12-wk old pigs for 4.5 h. In endotoxemic pigs, the phase 1 (i.e., 0-2 h) increases in pulmonary arterial pressure, pulmonary vascular resistance (PVR), and alveolar-arterial O2 gradient and the decreases in cardiac index (CI) and lung dynamic compliance (Cdyn) were blocked by NSAID. Thus phase 1 changes were cyclooxygenase dependent. Furthermore, these effects were blocked or greatly attenuated by DEX. During phase 2 of endotoxemia (i.e., 2-4.5 h), the increased PVR and decreased CI and Cdyn were not blocked by NSAID but were attenuated by DEX, suggesting the presence of cyclooxygenase-independent metabolites. Both NSAID and DEX blocked the endotoxin-induced increases in lung water, bronchoalveolar lavage (BAL) neutrophil, and BAL albumin content. The fall in plasma proteins persisted in NSAID but not DEX-treated pigs. We conclude that endotoxemia in the pig causes severe acute respiratory failure largely mediated by cyclooxygenase and possibly lipoxygenase products of arachidonic acid metabolism.     

Effect of porcine respiratory coronavirus infection on lipopolysaccharide recognition proteins and haptoglobin levels in the lungs

Porcine respiratory coronavirus (PRCV) potentiates respiratory disease and proinflammatory cytokine production in the lungs upon intratracheal inoculation with lipopolysaccharide (LPS) at 1 day of infection. This study aimed to quantify LPS-binding protein (LBP), CD14 and haptoglobin in the lungs throughout a PRCV infection. LBP and CD14 recognize LPS and enhance its endotoxic activity, whereas haptoglobin dampens it. Gnotobiotic pigs were inoculated intratracheally with PRCV (n = 34) or saline (n = 5) and euthanized 1-15days post inoculation (DPI). Virus was detected in the lungs from 1 to 9DPI. Cell-associated CD14 in lung tissue increased up to 15 times throughout the infection, due to an increase in highly CD14+ monocyte-macrophages from 1 to 12DPI and CD14+ type 2 pneumocytes from 7 to 9DPI. LBP and soluble CD14 levels in bronchoalveolar lavage fluids were elevated from 1-12DPI, with up to 35- and 4-fold increases, respectively. Haptoglobin levels increased significantly (x4.5) at 7DPI. In addition, we found that PRCV could sensitize the lungs to LPS throughout the infection, but the response to LPS appeared less enhanced at the end of infection (7DPI). The marked increases in LBP, CD14 and haptoglobin were not correlated with the extent of the LPS response.     

IFN-gamma-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma

We investigated the effects of IFN-gamma-inducing factor (IL-18) in a ragweed (RW) mouse model of allergic asthma. Administration of IL-18 in conjunction with allergic sensitization and challenge in wild-type, but not IFN-gamma -/- mice, inhibited the bronchoalveolar lavage (BAL) eosinophilia induced by RW challenge, and increased serum levels of RW-specific IgG2a and production of IFN-gamma from splenocytes cultured with RW, indicating a critical role for IFN-gamma in mediating these effects. Paradoxically, the same treatment schedule in WT mice increased serum levels of RW-specific IgE and IgG1, and production of IL-4 and IL-5 from splenocytes cultured with RW. When the effects of the same IL-18 treatment schedule were allowed to mature for 3 wk, the inhibition of lung eosinophil recruitment was replaced by augmentation of lung eosinophil recruitment. In another experiment, IL-18 administered only with allergic sensitization increased BAL eosinophilia and lung expression of IL-5 and IFN-gamma, while IL-18 administered only with RW challenge decreased BAL eosinophilia and increased lung IFN-gamma expression, while lung expression of IL-5 remained unchanged. IL-18 administered without RW or adjuvant to naive mice increased total serum IgE levels. Finally, intrapulmonary administrations of IL-18 plus RW in naive mice dramatically increased Th2 cytokine production, IgE levels, eosinophil recruitment, and airway mucus, demonstrating induction of allergic sensitization. This is the first report demonstrating that IL-18 promotes a Th2 phenotype in vivo, and potently induces allergic sensitization. These results suggest that IL-18 may contribute to the pathogenesis of allergic asthma.     

Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease

Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8(+) to CD4(+)TCRalphabeta lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-gamma and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-gamma and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.     

Severe acute respiratory syndrome: clinical and laboratory manifestations

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease with significant morbidity and mortality. An epidemic in 2003 affected 8,098 patients in 29 countries with 774 deaths. The aetiological agent is a new coronavirus spread by droplet transmission. Clinical and general laboratory manifestations included fever, chills, rigor, myalgia, malaise, diarrhoea, cough, dyspnoea, pneumonia, lymphopenia, neutrophilia, thrombocytopenia, and elevated serum lactate dehydrogenase (LD), alanine aminotransferase (ALT) and creatine kinase (CK) activities. Treatment has been empirical; initial potent antibiotic cover, followed by simultaneous ribavirin and corticosteroids, with or without pulse high-dose methylprednisolone, have been used. The postulated disease progression comprises (1) active viral infection, (2) hyperactive immune response, and (3) recovery or pulmonary destruction and death. We investigated serum LD isoenzymes and blood lymphocyte subsets of SARS patients, and found LD1 activity as the best biochemical prognostic indicator for death, while CD3+, CD4+, CD8+ and natural killer cell counts were promising predictors for intensive care unit (ICU) admission. Plasma cytokine and chemokine profiles showed markedly elevated Th1 cytokine interferon (IFN)-gamma, inflammatory cytokines interleukin (IL)-1beta, IL-6 and IL-12, neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-gamma-inducible protein-10 (IP-10) for at least two weeks after disease onset, but there was no significant elevation of inflammatory cytokine tumor necrosis factor (TNF)-alpha and anti-inflammatory cytokine IL-10. Corticosteroid reduced IL-8, MCP-1 and IP-10 concentrations from 5-8 days after treatment. Measurement of biochemical markers of bone metabolism demonstrated significant but transient increase in bone resorption from Day 28-44 after onset of fever, when pulse steroid was most frequently given. With tapering down of steroid therapy, there was a decrease in bone resorption marker together with an increase in bone formation markers round Day 50, suggesting that some of the bone loss might be reversed. Our research studies on the chemical pathology and clinical immunology of SARS should have implications for the pathophysiology and therapy of this potentially lethal infection.     

Role for innate IFNs in determining respiratory syncytial virus immunopathology

Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1(-/-) mice that are incapable of generating Th1 CD4(+) cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-alphabetaR (IFN-alphabetaR(-/-)), the IFN-gammaR (IFN-gammaR(-/-)), or both receptors (IFN-alphabetagammaR(-/-)). Although both the IFN-gammaR(-/-) and the IFN-alphabetagammaR(-/-) animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8(+) T cells could be detected in lungs of IFN-alphabetagammaR(-/-) animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4(+) Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.     

Differential cytokine mRNA expression in Dermatophagoides farinae allergen-sensitized and respiratory syncytial virus-infected mice

The interaction between mite allergen sensitization and respiratory syncytial virus (RSV) infection at the level of cytokine mRNA expression was examined in a murine model in the present study. Primary RSV infection enhances expression of inflammatory cytokines such as IL-6, IFN-gamma, and eotaxin in the lung and upregulates the expression of Th2-like cytokines IL-10 and IL-13 in the spleen in BALB/c mice. Mite antigen-sensitized and RSV-infected (ASRSV) mice show enhanced (P < 0.05) total serum IgE compared to antigen-sensitized mice. However, the levels of viral mRNA in the lung tissues are comparable between RSV-infected and ASRSV mice. It is concluded that compartmentalization of cytokine expression following RSV infection plays a role in the augmentation of Th2-like and IgE antibody response to RSV.     

Immune Modulatory Effects of IL-22 on Allergen-Induced Pulmonary Inflammation

IL-22 is a Th17/Th22 cytokine that is increased in asthma. However, recent animal studies showed controversial findings in the effects of IL-22 in allergic asthma. To determine the role of IL-22 in ovalbumin-induced allergic inflammation we generated inducible lung-specific IL-22 transgenic mice. Transgenic IL-22 expression and signaling activity in the lung were determined. Ovalbumin (OVA)-induced pulmonary inflammation, immune responses, and airway hyperresponsiveness (AHR) were examined and compared between IL-22 transgenic mice and wild type controls. Following doxycycline (Dox) induction, IL-22 protein was readily detected in the large (CC10 promoter) and small (SPC promoter) airway epithelial cells. IL-22 signaling was evidenced by phosphorylated STAT3. After OVA sensitization and challenge, compared to wild type littermates, IL-22 transgenic mice showed decreased eosinophils in the bronchoalveolar lavage (BAL), and in lung tissue, decreased mucus metaplasia in the airways, and reduced AHR. Among the cytokines and chemokines examined, IL-13 levels were reduced in the BAL fluid as well as in lymphocytes from local draining lymph nodes of IL-22 transgenic mice. No effect was seen on the levels of serum total or OVA-specific IgE or IgG. These findings indicate that IL-22 has immune modulatory effects on pulmonary inflammatory responses in allergen-induced asthma.     

CD4(+) T cell frequencies and Th1/Th2 cytokine patterns expressed in the acute and memory response to respiratory syncytial virus I-E(d)-restricted peptides

The respiratory syncytial virus (RSV)-specific frequencies and cytokine expression patterns of acute and memory CD4(+) T cells from RSV strain-A- and strain-B-infected BALB/c mice were determined following restimulation with a panel of 14 predicted RSV I-E(d) peptides from NSP-2, M, SH, F, and L proteins. Ten of fourteen peptides stimulated intracellular Th1 and/or Th2 cytokines in CD4(+) T cells from the mediastinal lymph nodes (MLN) and spleens of RSV strain-A- or strain-B-immune BALB/c mice. Spleen cells exhibited a predominant Th2 cytokine expression pattern after peptide stimulation, whereas MLN cells exhibited a mixed Th1/Th2 cytokine pattern. For a few peptides, there were differences in the Th1/Th2 cytokine response to peptides from the homologous versus heterologous RSV group. None of the 10 peptides induced both Th1 and Th2 cytokines in cells from similarly immunized mice. The frequency and breadth of cytokine expression by I-E(d)-restricted CD4(+) T cells to peptide stimulation was diminished in the memory response.     

Dexamethasone blocks increased leukotriene B4 production during endotoxin-induced lung injury

We hypothesized that leukotriene B4 (LTB4) might be produced during endotoxemia in pigs and, if so, might play a role in the pathophysiology of acute respiratory failure. Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3 h. Endotoxemic pigs were treated with dexamethasone (DEX, iv) 18 h (5 mg/kg) and 1 h (5 mg/kg) before onset of endotoxemia. During phases I (i.e., 0-2 h) and II (i.e., 2-4 h), endotoxin decreased cardiac index, caused granulocytopenia, and increased mean pulmonary arterial pressure, pulmonary vascular resistance, alveolar-arterial O2 gradient, and hematocrit. During phase II, plasma LTB4 levels were increased (as determined by radioimmunoassay, reverse-phase high-performance liquid chromatography, and ultraviolet spectroscopy). Endotoxin increased the levels of LTB4 and albumin in bronchoalveolar lavage fluid (BALF). DEX blocked or greatly attenuated the endotoxin-induced hemodynamic abnormalities and blocked the increases in plasma and BALF LTB4 levels. We conclude that LTB4 is produced during porcine endotoxemia and could possibly play a role in the pathophysiology of endotoxin-induced lung injury in anesthetized pigs.     

Repeated respiratory Mycoplasma pneumoniae infections in mice: effect of host genetic background

Respiratory Mycoplasma pneumoniae (Mp) infection is involved in several acute and chronic lung diseases including community-acquired pneumonia, asthma and chronic obstructive pulmonary disease. In the chronic disease process, recurrent respiratory bacterial infections could occur, which may result in varying degrees of symptoms and lung inflammation among patients. However, the lung immunologic differences of host responses to repeated bacterial (i.e., Mp) infections remain to be determined. In the present study, we examined cellular and humoral responses to multiple (up to 3) Mp infections in two genetically different strains of mice (BALB/c and C57BL/6). Mice were intranasally inoculated with one Mp infection, two or three Mp infections (4 weeks apart), and sacrificed on days 3, 7 and 14 after the last Mp infection. Overall, compared to C57BL/6 mice, BALB/c mice demonstrated a significantly higher degree of lung tissue inflammatory cell infiltrate, BAL cellularity, and release of pro-inflammatory cytokines (TNF-alpha, keratinocyte-derived chemokine (KC, a mouse homolog of human chemokine Gro-alpha [CXCL1], and IFN-gamma). In addition, BALB/c mice presented higher levels of serum Mp-specific IgG and IgM, but not IgA. Consistently with lung and serum data, Mp load in BAL and lung specimens was significantly higher in BALB/c mice than C57BL/6 mice. Moreover, repeated Mp infections in BALB/c, but not C57BL/6 mice, produced a greater inflammatory response than did a single Mp infection. Our results suggest that hosts with different genetic background may have different susceptibility to repeated respiratory Mp infections along with inflammatory responses.     

IL-12p40 and IL-18 modulate inflammatory and immune responses to respiratory syncytial virus infection

Respiratory syncytial virus-induced bronchiolitis has been linked to the development of allergy and atopic asthma. IL-12 and possibly IL-18 are central mediators orchestrating Th1 and/or Th2 immune responses to infection. To determine a possible role for IL-12 in regulating the immune response to acute respiratory syncytial virus infection, IL-12p40 gene-targeted (IL-12p40-/-) and wild-type mice were intratracheally infected with respiratory syncytial virus, and lung inflammatory and immune responses were assessed. Lung inflammation and mucus production were increased in the airways of IL-12p40-/- mice as compared with those of wild-type mice, concurrent with increased levels of the Th2 effector cytokines IL-5 and IL-13. Respiratory syncytial virus clearance and levels of Th1 effector cytokine IFN-gamma were not altered. Interestingly, IL-18, another mediator of IFN-gamma production, was significantly increased in the lungs of IL-12p40-/- mice early during the course of infection. Abrogation of IL-18-mediated signaling in IL-12p40-/- mice further enhanced Th2 immune response and mucus production in the airways during respiratory syncytial virus infection but failed to modulate IFN-gamma production or viral clearance. These findings implicate a role for IL-12 and IL-18 in modulating respiratory syncytial virus-induced airway inflammation distinct from that of viral clearance.     

Prolonged exercise suppresses antigen-specific cytokine response to upper respiratory infection.

Fatiguing exercise has been associated with an increased susceptibility to infection. This study examined the antigen-specific T-helper (Th) type 1 and Th type 2 cytokine response to herpes simplex virus (HSV) infection after an acute bout of fatiguing exercise. Male BALB/cJ mice ran on a treadmill (Ex) until voluntary fatigue (approximately 2.5 h), and control mice were handled and remained next to the treadmill. Mice were infected with HSV 20 min after exercise. Mice were killed 2 or 7 days postinfection, and sera and spleens were taken for the determination of HSV-specific serum IgM, splenocyte cytokine production during culture with HSV, and splenocyte natural killer cell cytotoxicity. Both Th type 1 [interleukin (IL)-2, interferon-gamma, IL-12] and Th type 2 (IL-10) cytokine production in spleen cell cultures, as well as natural killer cell cytotoxicity, decreased in Ex on day 2 postinfection. On day 7 postinfection, there was no difference in HSV-specific serum IgM or cytokine production by cells from control and Ex mice, with the exception of decreased IL-12 in Ex mice. These findings suggest that fatiguing exercise may alter the kinetics of antigen-specific cytokine production.     

Amniotic Fluid Stem Cells Inhibit the Progression of Bleomycin-Induced Pulmonary Fibrosis via CCL2 Modulation in Bronchoalveolar Lavage

The potential for amniotic fluid stem cell (AFSC) treatment to inhibit the progression of fibrotic lung injury has not been described. We have previously demonstrated that AFSC can attenuate both acute and chronic-fibrotic kidney injury through modification of the cytokine environment. Fibrotic lung injury, such as in Idiopathic Pulmonary Fibrosis (IPF), is mediated through pro-fibrotic and pro-inflammatory cytokine activity. Thus, we hypothesized that AFSC treatment might inhibit the progression of bleomycin-induced pulmonary fibrosis through cytokine modulation. In particular, we aimed to investigate the effect of AFSC treatment on the modulation of the pro-fibrotic cytokine CCL2, which is increased in human IPF patients and is correlated with poor prognoses, advanced disease states and worse fibrotic outcomes. The impacts of intravenous murine AFSC given at acute (day 0) or chronic (day 14) intervention time-points after bleomycin injury were analyzed at either day 3 or day 28 post-injury. Murine AFSC treatment at either day 0 or day 14 post-bleomycin injury significantly inhibited collagen deposition and preserved pulmonary function. CCL2 expression increased in bleomycin-injured bronchoalveolar lavage (BAL), but significantly decreased following AFSC treatment at either day 0 or at day 14. AFSC were observed to localize within fibrotic lesions in the lung, showing preferential targeting of AFSC to the area of fibrosis. We also observed that MMP-2 was transiently increased in BAL following AFSC treatment. Increased MMP-2 activity was further associated with cleavage of CCL2, rendering it a putative antagonist for CCL2/CCR2 signaling, which we surmise is a potential mechanism for CCL2 reduction in BAL following AFSC treatment. Based on this data, we concluded that AFSC have the potential to inhibit the development or progression of fibrosis in a bleomycin injury model during both acute and chronic remodeling events.     

Th1-Th2 cytokine kinetics in the bronchoalveolar lavage fluid of mice infected with Cryptococcus neoformans of different virulences

Th1 immune response plays an important role in protection against infection with Cryptococcus neoformans in mice. We investigated the effect of virulence of C. neoformans on cytokine production in the lung of a mouse model of pulmonary cryptococcosis. BALB/c mice were inoculated intratracheally with a high or low virulence strain of C. neoformans, followed by serial measurements of Th1 and Th2 cytokine concentrations in the bronchoalveolar lavage (BAL) fluid using appropriate enzyme-linked immunosorbent assay kits. The number of colony-forming units (CFU) increased with time, and all mice infected with the highly virulent strain were dead at 28 days after inoculation. In contrast, the number of microorganisms diminished with time in the mice infected with the low virulence strain during the 4-week study. The numbers of neutrophils and lymphocytes in the BAL fluid paralleled those of CFU. High neutrophil counts were observed in the BAL fluid of mice infected with the highly virulent strain, while lymphocyte counts were increased only in the later part of the study in mice infected with the high and low virulence strains. The concentrations of Th2 cytokine, interleukin (IL)-4 were significantly higher in mice infected with the highly virulent strain at days 14 and 21 of infection, whereas the level of Th1 cytokine, interferon-gamma, was significantly higher in the latter strain at days 7 and 14. Our results suggest that strain-specific difference in the organism's ability to induce (or evade) the host immune system contributes to the outcome of infection.