Animal and translational models of SARS-CoV-2 infection and COVID-19.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

The genetic control of susceptibility to Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of the most successful human pathogens, surviv ing in latent foci of infection in one third of humanity, yet causing lung necrosis in sufficient individuals to ensure its transmission. Each stage of the host response to M. tuberculosis is under genetic control, including the initial encounter with mycobacteria by macrophages, epithelial cells and dendritic cells in the lung, induction of the inductive T cell response, and killing by activated macrophages within granulomas. Although environmental factors are important determinants of progression to disease, there is a genetic component underlying susceptibility to tuberculosis (TB), the basis of which may vary in different populations. Recent studies using a variety of methods have defined a number of susceptibility alleles for the development of active TB. Many of these influence macrophage responses to mycobacteria. We have studied the influence of loss of function polymorphisms in the human P2X7 gene on the capacity of macrophages to kill M. tuberculosis. Activation of the P2X7 receptor, an ATP-gated Ca2+ channel, leads to the formation of pores, the activation of phospholipase D, and the induction of apoptosis with death of the infecting mycobacteria. Macrophages from subjects who are heterozygote, homozygote or compound heterozygote for these polymorphisms fail to undergo apoptosis and show partial or complete inhibition of mycobacterial killing. One of these non-functioning polymorphisms was significantly associated with increased susceptibility to TB disease, particularly extrapulmonary disease, in two unrelated cohorts of TB patients. Insights into the genetic regulation of susceptibility to human TB may identify novel methods for controlling latent M. tuberculosis and reducing the burden of tuberculosis.

Independent protective effects for tumor necrosis factor and lymphotoxin alpha in the host response to Listeria monocytogenes infection.

Although the essential role of tumor necrosis factor (TNF) in resistance to Listeria monocytogenes infection is well established, the roles of the related cytokines lymphotoxin alpha (LTalpha) and lymphotoxin beta (LTbeta) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTalpha, and LTbeta in the host response to Listeria. To overcome the lack of peripheral lymph nodes in LTalpha(-/-) and LTbeta(-/-) mice, bone marrow chimeras were constructed. TNF(-/-) and LTalpha(-/-) chimeras that lacked both secreted LTalpha(3) and membrane-bound LTalpha(1)beta(2) and LTalpha(2)beta(1) were highly susceptible and succumbed 4.5 and 6 days, respectively, after a low-dose infection (200 CFU). LTbeta(-/-) chimeras, which lacked only membrane-bound LT, controlled the infection in a manner comparable to wild-type (WT) chimeras. The Listeria-specific proliferative and gamma interferon T-cell responses were equivalent in all five groups of infected mice (LTalpha(-/-) and LTbeta(-/-) chimeras, WT chimeras, and TNF(-/-) and WT mice). TNF(-/-) mice and LTalpha(-/-) chimeras, however, failed to generate the discrete foci of lymphocytes and macrophages that are essential for bacterial elimination. Rather, aberrant necrotic lesions comprised predominantly of neutrophils with relatively few lymphocytes and macrophages were observed in the livers and spleens of TNF(-/-) and LTalpha(-/-) chimeras. Therefore, in addition to TNF, soluble LTalpha(3) plays a separate essential role in control of listerial infection through control of leukocyte accumulation and organization in infected organs.

T cell-derived tumour necrosis factor is essential, but not sufficient, for protection against Mycobacterium tuberculosis infection.

Tumour necrosis factor (TNF) is critical for sustained protective immunity against Mycobacterium tuberculosis infection. To investigate the relative contributions of macrophage- and T cell-derived TNF towards this immunity T cells from wild-type (WT) or TNF-/- mice were transferred into RAG-/- or TNF-/- mice which were then infected with M. tuberculosis. Infected RAG-/- mice and RAG-/- recipients of TNF deficient T cells developed overwhelming infection, with extensive pulmonary and hepatic necrosis and succumbed with a median of only 16 days infection. By contrast, RAG-/- recipients of WT T cells showed a significant increase in survival with a median of 32 days. Although initial bacterial growth was similar in all groups of RAG-/- mice, the transfer of WT, but not TNF-/-, T cells led to the formation of discrete foci of leucocytes and macrophages and delayed the development of necrotizing pathology. To determine requirements for macrophage-derived TNF, WT or TNF-/- T cells were transferred into TNF-/- mice at the time of M. tuberculosis infection. Transfer of WT T cells significantly prolonged survival and reduced the early tissue necrosis evident in the TNF-/- mice, however, these mice eventually succumbed indicating that T cell-derived TNF alone is insufficient to control the infection. Therefore, both T cell- and macrophage-derived TNF play distinct roles in orchestrating the protective inflammatory response and enhancing survival during M. tuberculosis infection.

Immunological basis for reactivation of tuberculosis in mice.

In this study different inbred strains of mice appeared to control and contain a low dose aerosol infection with Mycobacterium tuberculosis in a similar manner, giving rise to a chronic state of disease. Thereafter, however, certain strains gradually began to show evidence of regrowth of the infection, whereas others consistently did not. Using C57BL/6 mice as an example of a resistant strain and CBA/J mice as an example of a strain susceptible to bacterial growth, we found that these animals revealed distinct differences in the cellular makeup of lung granulomas. The CBA/J mice exhibited a generally poor lymphocyte response within the lungs and vastly increased degenerative pathology at a time associated with regrowth of the infection. As a possible explanation for these events, it was then observed that the CBA/J mouse strain was also less able to upregulate adhesion molecules, including CD11a and CD54, on circulating lymphocytes. These results therefore suggest that a failure to control a chronic infection with M. tuberculosis may reflect an inability to localize antigen-specific lymphocytes within the lung.

Restraining mycobacteria: role of granulomas in mycobacterial infections.

The generation of prolonged immunity to Mycobacterium tuberculosis requires not only an antigen-specific IFN-gamma-producing T cell response, including both CD4 and CD8 T cells, but also the generation of protective granulomatous lesions, whereby the close apposition of activated T cells and macrophages acts to contain bacterial growth. The importance of the granulomatous lesion in controlling this immune response and in limiting both tissue damage and bacterial dissemination has been considered a secondary event but, as the present review illustrates, is no less important in surviving mycobacterial infection than an antigen-specific T-cell response. The formation of a protective granuloma involves the orchestrated production of a host of chemokines and cytokines, the upregulation of their receptors along with upregulation of addressins, selectins and integrins to coordinate the recruitment, migration and retention of cells to and within the granuloma. In the present review, the principal components of the protective response are outlined and the role of granuloma formation and maintenance in mediating prolonged containment of mycobacteria within the lung is addressed.

Interleukin-6 induces early gamma interferon production in the infected lung but is not required for generation of specific immunity to Mycobacterium tuberculosis infection.

Immunity to Mycobacterium tuberculosis is dependent upon the generation of a protective gamma interferon (IFN-gamma)-producing T-cell response. Recent studies have suggested that interleukin-6 (IL-6) is required for the induction of a protective T-cell response and that IL-4 may suppress the induction of IFN-gamma. To evaluate the role of the cytokines IL-6 and IL-4 in the generation of pulmonary immunity to M. tuberculosis, IL-6 and IL-4 knockout mice were infected with M. tuberculosis via aerosol. The absence of IL-6 led to an early increase in bacterial load with a concurrent delay in the induction of IFN-gamma. However, mice were able to contain and control bacterial growth and developed a protective memory response to secondary infection. This demonstrates that while IL-6 is involved in stimulating early IFN-gamma production, it is not essential for the development of protective immunity against M. tuberculosis. In contrast, while the absence of IL-4 resulted in increased IFN-gamma production, this had no significant effect upon bacterial growth.

Neutrophils play a protective nonphagocytic role in systemic Mycobacterium tuberculosis infection of mice.

Evidence showing that neutrophils play a protective role in the host response to infection by different intracellular parasites has been published in the past few years. We assessed this issue with regard to the infection of mice with Mycobacterium tuberculosis. We found a chronic recruitment of neutrophils to the infection foci, namely, to the peritoneal cavity after intraperitoneal infection and to the spleen and liver after intravenous inoculation of the mycobacteria. However, bacilli were never found associated with the recruited neutrophils but rather were found inside macrophages. The intravenous administration of the antineutrophil monoclonal antibody RB6-8C5 during the first week of infection led to selective and severe neutropenia associated with an enhancement of bacillary growth in the target organs of the mice infected by the intravenous route. The neutropenia-associated exacerbation of infection was most important in the liver, where a bacterial load 10-fold higher than that in nonneutropenic mice was found; the exacerbation in the liver occurred both during and after the neutropenic period. Early in infection by M. tuberculosis, neutropenic mice expressed lower levels of mRNAs for gamma interferon and inducible nitric oxide synthase in the liver compared to nondepleted mice. These results point to a protective role of neutrophils in the host defense mechanisms against M. tuberculosis, which occurs early in the infection and is not associated with the phagocytic activity of neutrophils but may be of an immunomodulatory nature.

Granuloma formation is required to contain bacillus growth and delay mortality in mice chronically infected with Mycobacterium tuberculosis.

Previous studies in this laboratory have shown that mice with a gene disruption to the intracellular adhesion molecule-1 (ICAM-K/O) express normal cell-mediated immunity but cannot mount delayed-type hypersensitivity reactions following Mycobacterium tuberculosis infection. However, even in the absence of any appreciable granuloma formation, these mice control bacterial growth for at least 90 days. While not required to control the infection initially, we hypothesized that granuloma formation was required to control chronic infection, acting by surrounding infected cells to prevent bacterial dissemination. To test this, ICAM-1 knockout mice were infected with a low dose aerosol of M. tuberculosis Erdman and were found to succumb to infection 136+/-30 days later, displaying highly elevated bacterial loads compared to wild-type mice. Lung tissue from ICAM-K/O mice displayed extensive cellular infiltration and widespread tissue necrosis, but no organized granulomatous lesions were evident, whereas the control mice displayed organized compact granulomas. These data demonstrate that while a granulomatous response is not required initially to control M. tuberculosis infection, absence of granulomas during chronic infection leads to increased bacterial growth and host death. Thus these data support the hypothesis that granuloma formation is required to control chronic infection, acting by surrounding and walling off sites of infection to prevent bacterial dissemination and maintain a state of chronic infection.

Role of gamma delta T cells in immunopathology of pulmonary Mycobacterium avium infection in mice.

Several studies have shown that gamma delta T cells influence granuloma development after infection with intracellular pathogens. The role of gamma delta T cells in controlling the influx of inflammatory cells into the lung after Mycobacterium avium infection was therefore examined with gene-disrupted mice (K/O). The mice were infected with either M. avium 724, a progressively replicating highly virulent strain of M. avium, or with M. avium 2-151 SmT, a virulent strain that induces a chronic infection. gamma delta-K/O mice infected with M. avium 2-151 SmT showed early enhanced bacterial growth within the lung compared to the wild-type mice, although granuloma formation was similar in both strains. gamma delta-K/O mice infected with M. avium 724 showed identical bacterial growth within the lung compared to the wild-type mice, but they developed more-compact lymphocytic granulomas and did not show the extensive neutrophil influx and widespread tissue necrosis seen in wild-type mice. These data support the hypothesis that isolates of M. avium that induce protective T-cell-specific immunity are largely unaffected by the absence of gammadelta T cells. Whereas with bacterial strains that induce poor protective immunity, the absence of gamma delta T cells led to significant reductions in both the influx of neutrophils and tissue damage within the lungs of infected mice.

Intranasal infection of beige mice with Mycobacterium avium complex: role of neutrophils and natural killer cells.

Beige mice show increased susceptibility to intranasal infection with organisms of the Mycobacterium avium complex (MAC) compared with their immunocompetent congenics, C57BL/6 mice. This increased susceptibility was clear 2 weeks postinfection, before the activation of the specific immune response. T lymphocytes from 4-week infected beige mice, cultured in vitro, produced amounts of gamma interferon similar to those found in cells from C57BL/6 mice. Macrophage activation, as judged by NO production and lysis of the macrophage target P815, occurred in the lungs of beige mice. Despite the inability of bone marrow-derived NK cells from beige mice to lyse NK-susceptible YAC-1 cells, their gamma interferon production was normal. Monoclonal antibody to NK1.1 was used to deplete C57BL/10 mice of lytic activity against YAC-1 cells without exacerbating infection between 2 and 6 weeks of observation, making it unlikely that any deficiency in NK cells was the cause of susceptibility in beige mice. There was a striking influx of neutrophils in the lungs of beige mice compared with C57BL/6. More than half of the MAC organisms appeared associated with the neutrophils of beige mice, while in C57BL/6 mice, most MAC organisms were associated with cells of macrophage/monocyte morphology. Injection of monoclonal antibody specific for neutrophils failed to eliminate those cells from the lungs of beige mice. However, in C57BL/6 mice, neutrophil numbers were reduced by 95% without exacerbating the infection. We conclude that, although neutrophils are not essential to the relative resistance of C57BL/6 mice, the known deficiencies in both neutrophils and macrophages account for the susceptibility of beige mice.

Increased lung cell cytotoxic but not bactericidal or phagocytic activity in Mycobacterium avium complex-infected mice.

Following intranasal infection of mice with Mycobacterium avium complex (MAC) organisms, bacterial growth plateaued at the fourth week postinfection and then remained relatively constant thereafter. Inflammatory cell numbers in the lungs increased 10-fold by 4 weeks postinfection, and lung cell cytotoxicity and the production of NO, H202, and 02- by lung cell cultures had all increased significantly by this time and remained elevated throughout the 15-week experimental study. Although these parameters are generally associated with increased bactericidal activity, there appeared to be a defect in phagocytosis by lung cells, so that bactericidal activity could not be demonstrated in either in vitro or in vivo experiments. This study suggests that following intranasal infection with MAC, inflammatory cells are activated, sufficient to prevent further bacterial growth in vivo but not sufficient to clear the infection. We suggest that the deficiency may lie in the phagocytic activity of the cells.

Endogenous interleukin-12 is involved in resistance of mice to Mycobacterium avium complex infection.

Acquired cellular resistance against Mycobacterium avium complex (MAC) infections involves the induction of Th1 type gamma interferon (IFN-gamma)-producing T cells. Interleukin-12 (IL-12) is a cytokine involved in the control of IFN-gamma production by T cells and NK cells. The role of IL-12 in the response to MAC infection was investigated. Depletion of endogenous IL-12 by injection of monoclonal antibody prior to and during intranasal infection with MAC resulted in an 150- to 550-fold increase in bacterial load in lung, spleen, and liver homogenates by 10 weeks postinfection. Depletion of IL-12 abrogated the ability of spleen cell cultures to produce IFN-gamma in response to stimulus with live MAC. IL-12-depleted mice showed a 75% decrease in the number of inflammatory cells entering the lungs following intranasal infection with MAC, with significant reductions in cytotoxic activity and nitric oxide production by lung cells. This work suggests that IL-12 plays a major role in the activation of IFN-gamma-producing cells during MAC infection.

Inflammatory response following intranasal infection with Mycobacterium avium complex: role of T-cell subsets and gamma interferon.

The role of CD4+ and CD8+ T cells in the response to intranasal infection with a Mycobacterium avium complex isolate (MAC) was investigated. Depletion of CD4+ T cells by injected antibody exacerbated infection in the lung, spleen, and liver. There were decreased numbers of inflammatory cells in the lungs of CD4-depleted mice and a significant decrease in lung cytotoxic activity. The neutrophil response was unaffected, and in CD4-depleted mice, unlike intact infected mice, these cells were found with large numbers of associated MAC. Purified CD4+ splenic T cells produced gamma interferon (IFN-gamma) in vitro in response to MAC antigen. IFN-gamma production by cultured spleen, lung, or mediastinal lymph node cells was markedly reduced in CD4-depleted mice. In contrast, CD8+ T cells did not produce IFN-gamma in vitro, and depletion of CD8+ T cells from infected mice had no effect on bacterial growth or lung cell activation. Depletion of IFN-gamma by injected monoclonal antibody had effects similar to those of CD4 depletion, namely, exacerbation of infection and decreased lung cell cytotoxicity. We conclude that CD4+ T cells are the main T cells involved in the lung response to MAC infection and that this response is at least partially dependent on the production of IFN-gamma.

Interleukin-6 production during chronic experimental infection.

The appearance of interleukin-6 (IL-6) in serum of mice was monitored during the course of chronic infection with either Brucella abortus vaccine strain 19 or a virulent Mycobacterium avium Complex (MAC) isolate. Serum IL-6 during brucella infection was higher than during infection with MAC, despite similar numbers of bacteria. Furthermore, IL-6 titres decreased after the peak of infection, falling to baseline levels before these chronic infections were eradicated. The ability of peritoneal cells or spleen cell suspensions to produce IL-6 under either specific or non-specific stimulus was greatly enhanced by infection. While production of IL-6 by these cultures was apparently mostly independent of T cells, T cells from infected mice could produce an IL-6 response. Thus CD4+ T lymphocytes prepared from mice which had recovered from B. abortus infection, cultured with antigen and antigen presenting cells, resulted in IL-6 production, which was not observed in similarly cultured CD8+ T cells, indicating a role for T cells.