Novelty in the gut: a systematic review and meta-analysis of the gastrointestinal manifestations of COVID-19.

BACKGROUND: The COVID-19 epidemic has affected over 2.6 million people across 210 countries. Recent studies have shown that patients with COVID-19 experience relevant gastrointestinal (GI) symptoms. We aimed to perform a systematic review and meta-analysis on the GI symptoms of COVID-19. METHODS: A literature search was conducted via electronic databases, including PubMed, Embase, Scopus, and Google Scholar, from inception until 20 March 2020. Data were extracted from relevant studies. A systematic review of GI symptoms and a meta-analysis comparing symptoms in severe and non-severe patients was performed using RevMan V.5.3. RESULTS: Pooled data from 2477 patients with a reverse transcription-PCR-positive COVID-19 infection across 17 studies were analysed. Our study revealed that diarrhoea (7.8%) followed by nausea and/or vomiting (5.5 %) were the most common GI symptoms. We performed a meta-analysis comparing the odds of having GI symptoms in severe versus non-severe COVID-19-positive patients. 4 studies for nausea and/or vomiting, 5 studies for diarrhoea and 3 studies for abdominal pain were used for the analyses. There was no significant difference in the incidence of diarrhoea (OR=1.32, 95% CI 0.8 to 2.18, Z=1.07, p=0.28, I2=17%) or nausea and/or vomiting (OR=0.96, 95% CI 0.42 to 2.19, Z=0.10, p=0.92, I2=55%) between either group. However, there was seven times higher odds of having abdominal pain in patients with severe illness when compared with non-severe patients (OR=7.17, 95% CI 1.95 to 26.34, Z=2.97, p=0.003, I2=0%). CONCLUSION: Our study has reiterated that GI symptoms are an important clinical feature of COVID-19. Patients with severe disease are more likely to have abdominal pain as compared with patients with non-severe disease.

High MafB expression following burn augments monocyte commitment and inhibits DC differentiation in hemopoietic progenitors.

We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC-II(+) population and mDCs (Gr1(neg)CD11b(+)CD11c(+)) with a corresponding decrease in bone marrow MHC-II(+) cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA-1, which is essential for DC development, but express high levels of MafB and M-CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mϕ and 1.6-fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC-II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M-CSFR expressions. Transient silencing of MafB (siRNA) in GMP-derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA-1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte-derived DC differentiation by regulating M-CSFR and GATA-1 expressions.

ß-adrenergic stimulation increases macrophage CD14 expression and E. coli phagocytosis through PKA signaling mechanisms.

CD14 is a glycoprotein that binds bacterial LPS in MØ. It is an essential component of the phagocytic system and is increased in septic shock. Critical injury and sepsis result in elevated endogenous CA levels. CAs have a significant impact on MØ inflammatory functions. We tested the hypothesis that ß-adrenergic stimulation regulates CD14 expression and bacterial phagocytosis in BMØ. Murine BMØ stimulated with isoproterenol (>8 h) induced a dose-dependent increase in cell surface CD14 expression. Specific PKA inhibitor (H-89) and gene-silencing (siRNA) studies demonstrated the role of cAMP-dependent PKA in mediating this response. In addition, we observed a correlation between an isoproterenol-mediated increase in CD14 expression and live Escherichia coli uptake in BMØ. Further, the essential role of CD14 in an isoproterenol-mediated increase in E. coli uptake was highlighted from experiments using CD14(-/-) mice. Moreover, the dose response of isoproterenol stimulation to CD14 expression and E. coli phagocytosis overlapped with similar EC50. Additionally, isoproterenol-mediated E. coli phagocytosis was prevented by H-89, suggesting that ß-adrenergic stimulus in BMØ increases CD14 expression and live E. coli phagocytosis through a common signaling pathway. Our studies indicate the potential impact of ß-adrenergic agents on important innate immune functions.

Propranolol restores the tumor necrosis factor-alpha response of circulating inflammatory monocytes and granulocytes after burn injury and sepsis.

Beta-adrenergic blockade ameliorates the hypermetabolism and catabolism in severe burn injury. Despite the salutary effects of beta-adrenergic blockade, the immunologic responses that accompany beta-blockade are not known. We have shown that burn sepsis is associated with increased sympathetic activation leading to altered monocytopoiesis and cytokine release in macrophages (MØ). Recent evidence suggests that murine MØ expressing F4/80+Gr1+ are the inflammatory phenotype. Here, we report that propranolol given after burn sepsis modulates the number and function of myeloid cells in circulation. B6D2F1 male mice were divided into sham (S), burn (B), and burn sepsis (BS) groups. Dorsal hair was shaved from S, B, and BS; B and BS received 15% scald burn; BS was inoculated with Pseudomonas Aeruginosa (PA 14, 4000-5000 colony-forming units) at the burn site. Mice from each group were then subjected to two different treatment regimens. One set received subcutaneous injections of propranolol (5 mg/kg body weight) at 24 and 48 hours after the injury while the control groups received saline. Blood was collected by cardiac puncture at 72 hours. The distribution of total F4/80+ monocyte population was determined by flow cytometry. Inflammatory monocyte subset was gated on Gr1+ expression in the F4/80+ fraction. Lipopolysaccharide-stimulated intracellular tumor necrosis factor (TNF)-alpha (ic-TNF) was also measured as an indicator of inflammatory response. The total F4/80+ monocyte fraction was significantly increased in BS (45 +/- 0.8%) vs S and B (10 +/- 0.8%; 9.5 +/- 0.6%). Propranolol treatment for 2 days reduced the number of circulating monocytes by 60% in BS. The mean fluorescent intensity (MFI) of ic-TNF produced per cell (F4/80+Gr1+ MØ) was significantly decreased in B and BS (S: 3043 +/- 213, B: 1638 +/- 343, BS: 1463 +/- 67). Of importance, propranolol treatment partially restored the MFI of ic-TNF (2177 +/- 114) and increased the percentage of inflammatory monocyte subset (F4/80+Gr1+) in BS by 70% compared with saline treatment. In contrast, beta-blockade after BS increased the percentage of granulocytes in circulation (28.4 +/- 3.6% in BS propranolol vs 15.4 +/- 0.3% in BS saline; P < .05) and augmented their TNF production (MFI = 903 +/- 102 in BS propranolol vs 644 +/- 5 in BS saline; P < .05). Propranolol reverses burn sepsis-induced monocytosis and simultaneously increases the number of granulocytes and enhances the inflammatory potential of the granulocytes and inflammatory monocyte subsets in circulation suggesting that monitoring MØ subsets and granulocytes in blood is a reliable biomarker to predict the efficacy of beta-blockade.

Perturbed bone marrow monocyte development following burn injury and sepsis promote hyporesponsive monocytes.

The mechanism of monocyte deactivation in critically injured burn patients remains unresolved. Two functionally distinct F4/80+Gr-1+ and F4/80+Gr-1- monocyte subsets have been characterized based on their homing to inflammatory or noninflammatory tissues, respectively. We hypothesized that the posttraumatic milieu in the bone marrow (BM) blunts the production of "inflammatory" monocytes. C57Blk/J male mice were divided into sham (S), burn (B), and burn sepsis (BS) groups. B and BS received a 15% dorsal scald burn and BS was inoculated with 15K colony forming units Pseudomonas aeruginosa at the burn site. Animals were killed and blood and femoral BM were collected 48, 72, and 96 hours after injury. ER-MP20 monocyte progenitors were isolated from BM and differentiated into macrophage (MØ) or dendritic cells (DCs) and characterized by the cell surface expression of F4/80 and CD11c, respectively. In both cell types, TLR-4 agonist induced cytokine levels were determined. Results showed a 2-fold increase in the F4/80+Gr-1+ subset at 48 hours in BS that started to decline at 72 hours and remained low at 96 hours. ER-MP20 progenitors isolated at 48 hours exhibited robust MØ differentiation potential but a significant decline in the percentage of the F4/80+Gr-1+ subset (P < .05 vs S) with a concomitant decrease in tumor necrosis factor alpha production. DC development from ER-MP20 progenitors and LPS-stimulated tumor necrosis factor alpha production were impaired. Therefore, BM progenitor derived MØ will replace the transient hyper-responsive circulating monocytes later during the course of the septic insult. Hypo-reactivity of the developing monocytes and DC in the BM and their subsequent egress to the periphery provide a plausible explanation for the immunosuppression that ensues a critical burn injury and sepsis.

Norepinephrine suppresses wound macrophage phagocytic efficiency through alpha- and beta-adrenoreceptor dependent pathways.

BACKGROUND: The systemic response to injury is characterized by massive release of norepinephrine (NE) into the circulation as a result of global sympathetic activation. We have recently demonstrated that NE modulates the recruitment of macrophages to the cutaneous wound. We hypothesized that NE suppresses wound macrophage phagocytic function through canonical adrenergic signaling pathways. METHODS: Murine wound macrophages were harvested at 5 days after injury and treated with physiologic and pharmacologic dose norepinephrine. Phagocytosis of green fluorescent protein-labeled Escherichia coli was assayed by flow cytometry. The signaling pathways mediating NE modulation of wound macrophage phagocytosis were interrogated by pharmacologic manipulation of alpha- and beta-adrenoreceptors (ARs), intracellular cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). Tissue specificity was determined by comparison of wound macrophages to splenic macrophages. RESULTS: Both physiologic and pharmacologic dose NE suppressed wound macrophage phagocytic efficiency. This effect was mediated by alpha- and beta-ARs in a dose-dependent fashion. Direct stimulation of cAMP-suppressed phagocytic efficiency and blockade of PKA signaling prevented NE-mediated suppression of phagocytic efficiency. Splenic macrophage phagocytic efficiency was less than that of wound macrophages and was not altered by NE. CONCLUSIONS: NE has a profound immunosuppressive effect on wound macrophage function that is tissue specific and appears to be mediated through adrenergic receptors and their canonical downstream signaling pathway. Attenuation of post-injury immunosuppression represents another potential mechanism by which beta-AR blockade may reduce morbidity and mortality after severe injury.

Severity of burn injury and sepsis determines the cytokine responses of bone marrow progenitor-derived macrophages.

BACKGROUND: Although thermal injury and sepsis result in enhanced monocytopoiesis, the functional characteristics of macrophages that develop in the microenvironment of burn and sepsis are unknown. Here we compare cytokine responses of bone marrow progenitor-derived macrophages (BMO) and peritoneal macrophages (PMO) after graded levels of thermal injury and sepsis. METHODS: Mice were randomly divided into sham (S), burn (B), and burn sepsis (BS) groups. The mild injury group received either a 7-second dorsal scald burn alone or in combination with 1,000 colony forming units (CFU) Pseudomonas aeruginosa at the wound site. The severe injury group was subjected to a 10-second burn with or without inoculation of 5,000 CFU P. aeruginosa. ER-MP12+ progenitors were separated from bone marrow cells 72 hour after injury. Macrophage colony stimulating factor (M-CSF) and Granulocyte-macrophage colony stimulating factor (GM-CSF) responsive clonogenic potentials, and lipopolysaccharide (LPS)-stimulated cytokine production were determined. RESULTS: In mild injury and sepsis, GM-CSF and M-CSF responsive clonal growth of ER-MP12+ progenitors was enhanced in the B and BS groups compared with the S group. M-CSF responsive colony growth in severe sepsis was significantly higher than that in all the other groups. LPS-stimulated tumor necrosis factor-alpha and Interleukin-6 levels were higher in the B and BS groups compared with the S group. Severe injury and sepsis attenuated this response significantly. The cytokine responses of PMO from both injury groups were similar to that of BMO. CONCLUSION: Severity of burn injury and the magnitude of sepsis influence the cytokine responses of BMO and PMO in a similar manner suggesting the microenvironment of burn injury and sepsis profoundly influence the functional phenotype of BMO.

Murine hematopoietic stem cells and progenitors express adrenergic receptors.

Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments monocytopoiesis and alters the beta-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of alpha1-, alpha2- and beta(2)-AR in enriched populations of ER-MP209(+) and ER-MP12(+) myeloid progenitors, CD117(+) and CD34(+) multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development.

A novel antibacterial gene transfer treatment for multidrug-resistant Acinetobacter baumannii-induced burn sepsis.

Sepsis caused by multidrug-resistant bacterial infections in critically injured patients has become a major clinical problem. Recently, Acinetobacter baumannii (AB) wound infections, especially in our critically injured soldiers fighting in Iraq and Afghanistan, is posing a major clinical problem and an economic burden. ConjuGon, Inc., has developed a novel antibacterial therapeutic technology using bacterial conjugation. The donor cells are attenuated Escherichia coli carrying a conjugative plasmid. The expression of bactericidal genes cloned on the plasmid is tightly repressed in the donor cells but becomes de-repressed once mobilized into a pathogen and disrupts protein synthesis. Here, we tested the efficacy of this novel conjugation technology to control and eradicate a drug-resistant clinical isolate of AB wound infection both in vitro and in a murine burn sepsis model. C57Blk/6J mice were divided into burn (B) and burn sepsis (BS) groups. All animals received a 12% TBSA dorsal scald full-thickness burn. The BS group was inoculated with multidrug-resistant AB (1 x 10(5) colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (10(3) - 19(10) CFU) of attenuated donor E. coli encoding bactericidal proteins. The survival rate was monitored for 10 days. The ability of donor cells to significantly diminish AB levels in the burn wound 24 hours after injury was determined by quantitative cultures. Donor cells were highly effective in killing AB in vitro. In the burn sepsis model, 90% B group animals survived, and 40% to 50% BS animals survived with no treatment in 5 to 6 days. Treatment with donor cells at 10(10) to 10(6) provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 x 10(4) CFU to 7.8 +/- 4.4 x 10(9) CFU in 24 hours in the untreated group. Single treatment with donor cells (10(10) CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 +/- 0.5 x 10(4) CFU; P < .05). Taken together, these results indicate that this novel technology is an efficient method to control drug-resistant AB burn wound infections and prevent their systemic spread.

Thermal injury and sepsis modulates beta-adrenergic receptors and cAMP responses in monocyte-committed bone marrow cells.

We have previously reported that adrenergic stimulation enhances monocytopoiesis following experimental burn injury and sepsis (BI/S). In the present work we measured beta-adrenergic receptor number and affinity in bone marrow committed monocyte progenitor cells (CD59(+)) following BI/S. We find that BI/S treatment significantly decreased monocyte progenitor cell beta-adrenergic receptors but significantly increased receptor binding affinity and isoproterenol-stimulated cAMP production. CD14 expression in macrophages derived in vitro from CD59(+) cells following BI/S was significantly increased by epinephrine and this change was blocked by beta(2)-adrenergic receptor antagonist. PCR analysis suggests the presence of beta(2)- but not beta(1)-adrenergic receptors. Enhanced adrenergic receptor signaling in CD59(+) bone marrow cells following BI/S may be important in macrophage development.

Adrenergic modulation of cytokine release in bone marrow progenitor-derived macrophage following polymicrobial sepsis.

Catecholamines may impact on the pathophysiology of sepsis by attenuating proinflammatory cytokine and augmenting antiinflammatory cytokine production by macrophages. We tested this premise in bone marrow monocyte progenitor-derived macrophages. Polymicrobial sepsis was induced in mice through cecal ligation and puncture. ER-MP 12 monocyte progenitors were isolated and differentiated into macrophages in vitro 72 hr later. Lipopolysaccharide (LPS)-stimulated cytokine production was measured with and without epinephrine, IL-10 and anti-IL-10 antibody. Epinephrine significantly increased IL-10 production, but attenuated TNF-alpha release exclusively through beta2 adrenergic receptors, and is independent of IL-10 production. Together, these results suggest that epinephrine can promote a potent antiinflammatory response in sepsis.

Adrenergic modulation of splenic macrophage cytokine release in polymicrobial sepsis.

Enhanced adrenergic stimulation and catecholamine release are important components of the pathophysiology of sepsis. Under physiological conditions, adrenergic stimulation has been shown to be a negative regulator of proinflammatory cytokine production through increasing IL-10 production. Here we have investigated if adrenergic stimulation similarly inhibits TNF-alpha and IL-6 production by splenic macrophages isolated from a polymicrobial sepsis model. Male B(6)D(2)F(1) mice were subjected to sham (S), laparotomy (Lap), and cecal ligation and puncture (CLP) under anesthesia. Splenic macrophages were isolated 72 h after the initial injury and were stimulated with endotoxin (LPS) in the presence and absence of epinephrine. Compared with S and Lap, splenic macrophages from the CLP group produced significantly less TNF-alpha and IL-6 and more IL-10 when stimulated with LPS. Macrophage cultures from CLP animals incubated with either epinephrine or IL-10 for 2 h had significantly reduced TNF-alpha and IL-6 release in response to LPS. However, similar cultures pretreated with IL-10 antibody before the addition of exogenous epinephrine failed to reverse the attenuation of LPS-stimulated cytokines. Pretreatment of macrophage cultures with beta(2)- (ICI-118551) but not beta(1)-adrenergic (atenolol) receptor antagonists reversed the epinephrine-mediated cytokine attenuation following LPS treatment. Data are also presented that demonstrate the involvement of protein kinase A activation with adrenergic agonist but not with IL-10 stimulation. Taken together, these findings suggest that adrenergic mechanisms may influence peripheral tissue macrophage inflammatory cytokine response following trauma and sepsis, independent of the effects of IL-10.

Burn injury and pulmonary sepsis: development of a clinically relevant model.

BACKGROUND: Despite improvements in the early resuscitation of the critically injured, mortality from multiple organ failure has remained stable, with the lung often the first organ to fail. Early intubation and mechanical ventilation predispose patients to the development of pneumonia and respiratory failure. Our objective was to establish a murine model of combined injury, consisting of burn/trauma and pulmonary sepsis with reproducible end-organ responses and mortality. METHODS: Male B6D2F1 mice were divided into four groups: burn/infection (BI), burn (B), infection (I), and sham (S). Burned animals had a full-thickness 15% dorsal scald burn. BI and I groups were inoculated intratracheally with Pseudomonas aeruginosa (3-5 x 103 colony-forming units). S and B animals received saline intratracheally. All animals were resuscitated with 2 mL of intraperitoneal saline. Mortality was recorded at 24, 48, and 72 hours. Bacterial sepsis was confirmed by tissue Gram's stain of the lungs and positive organ and blood cultures for Pseudomonas aeruginosa. Femoral bone marrow cells were collected at 72 hours from surviving animals. Clonogenic potential was assessed by response to macrophage (M) colony-stimulating factor (CSF) and granulocyte-macrophage (GM) CSF in a soft agar assay and the data were represented as colonies per femur. Isolated alveolar macrophages and whole lung tissue were assayed for levels of the inflammatory cytokines tumor necrosis factor-alpha and interleukin-6. RESULTS: Mortality at 72 hours was 30% in BI, 12% in I, and <10% in B and S groups. Pneumonia was documented in all infected animals at 24 hours by Gram's stain and positive tissue cultures for Pseudomonas aeruginosa. Systemic sepsis as confirmed by blood, and remote organ cultures was seen in BI animals only. Significantly increased responsiveness to M-CSF stimulations was noted in all groups (BI, 8,291 +/- 1,402 colonies/femur; B, 6,357 +/- 806 colonies/femur; and I, 8,054 +/- 1,112 colonies/femur; p < 0.05) relative to sham (3,369 +/- 883 colonies/femur, p < 0.05). Maximal responsiveness to GM-CSF stimulation was noted in the BI group (11,932 +/- 982 colonies/femur, p < 0.05), and similar GM responsiveness was noted in all other groups (B, 7,135 +/- 548 colonies/femur; I, 7,023 +/- 810 colonies/femur; and S, 6,829 +/- 1,439 colonies/femur). Alveolar macrophage release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 increased in all animals, but the magnitude of increase was not proportional to the strength of the inciting stimulus. CONCLUSION: Although minimal perturbations were seen after burn or pulmonary infection alone, the combined insult of burn and pulmonary sepsis resulted in statistically significant hematopoietic changes with increased monocytopoiesis. Only the combined injury resulted in systemic sepsis and significantly increased mortality. We have developed a clinically relevant model of trauma and pulmonary sepsis that will allow further clarification of the inflammatory response after injury and infection.