*2.1. Inflammation and Dysbiosis of the Elderly Patient*

The gut microbiota shows a great inter-individual variation and the human intestinal microbiome, i.e., the community that includes the genetic heritage and environmental interactions of all microorganisms, is very diverse and complex and continues to fluctuate during the various stages of life [43]. Furthermore, the intestinal microbiome is closely associated with various characteristics of integrity of the intestinal barrier, anti-inflammatory balance, immune and cardio-metabolic health, as well as the intestine–brain axis [41,44]. A loss of microbiota stability has been frequently observed in the elderly. The disruption of the intestinal barrier integrity as well as a condition of intestinal dysbiosis can further complicate the state of severity in SARS-CoV-2 [45,46]. In severe cases of SARS-CoV-2, elevated zonulin levels are a marker related to increased mortality. Measurement of LPS

binding protein, a marker of inflammation, revealed a significant increase in more severe cases, supporting the association between severe COVID-19 and loss of intestinal barrier integrity and microbial translocation [47].

The excessive accumulation of senescent cells present in aging and age-related diseases can contribute to chronic silent inflammation and tissue and organic dysfunction. Furthermore, old-age problems could contribute to a greater predisposition to various infectious and associated diseases of the intestine causing alterations in the microbiota of the elderly [30,48,49].

Studies have shown a decrease in the diversity of gut microbiota composition in the elderly, and COVID-19 patients manifest faecal microbiome alterations, with an increase in opportunistic pathogens and a depletion of commensal beneficial microorganisms [14,15]. Older people are known to have a less diverse gut microbiota and a noticeable decrease in beneficial microorganisms such as *Bifidobacterium.* Since diet, drug intake and the composition of the gut microbiome undergo substantial changes during aging, the intestinal metabolic environment, and therefore the levels of microbial metabolites, are influenced by age. Gut microbial-derived metabolites play a key role in inflammatory signalling by interacting with host immune cells. Some bacterial species, such as *Faecalibacterium prausnitzii*, *Roseburia intestinalis*, and *Anaerostipes butyraticus* are able to digest complex carbohydrates by fermentation, generating short-chain fatty acids (SCFA), fatty acids with fewer than six carbon atoms, consisting mainly of acetate, propionate and butyrate [32,50,51]. The elderly has lower SCFA levels than young subjects. A decrease in SCFA production in the colon has been linked to lower fibre intake and antibiotic treatments in the elderly by regulating expression of pro-inflammatory cytokines including IL-6, IL-12 and TNF-α [52]. SCFAs are important for their ability to reduce intestinal inflammation, protect against pathogenic invasion and maintain barrier integrity primarily by activating G-protein-coupled receptors (GPCRs) or inducing their suppressive effects on histone deacetylase (HDAC), and by affecting gene expression. The genesis of the cytokine storm could take place in the gastrointestinal tract [41]. SCFAs maintain the physiology of the intestinal epithelium by regulating cell turnover and barrier functions. SCFAs constitute a key regulatory system for the activation, recruitment and differentiation of immune cells, including neutrophils, macrophages, dendritic cells (DCs) and T lymphocytes [41,50]. A reduced abundance of butyrate-producing species is found in COVID-19 patients [51] and it has been suggested that the use of butyrate-producing species in COVID-19 patients in order to maintain the integrity of epithelium at the level of tight junctions could likely help reduce invasion of SARS-CoV-2 [52].

The intestinal microbiota plays a crucial role in gastrointestinal physiology by providing, among other activities, the synthesis of endogenous vitamins, such as vitamin K and most of the components of the vitamin B complex [41]. In particular, vitamin B constitutes an important support for the correct activation of the immune response, and interestingly, it improves respiratory function, maintains the integrity of the endothelium and prevents hypercoagulability. A dysbiotic condition could lead to a vitamin B deficiency and could significantly impair immune function. Therefore, B vitamins could be a crucial aid in the treatment of SARS-CoV-2 [52].

Another interesting aspect of diet in the elderly is the consumption of proteins, and in particular a diet that includes an excessive amount of proteins can be responsible for an increase in the intestinal production of potentially deleterious bacterial metabolites. The requirement for a higher dose of protein in elderly subjects is suggested in order to compensate for the lower sensitivity to anabolic stimulus. However, the very amount of protein in the diet and additional amino acids can influence the onset and progression of inflammation [53–55]. This condition could also, in turn, affect epithelial repair since some bacterial metabolites inhibit respiration of colon epithelial cells, cell proliferation and/or the influence of barrier function.

Another element that characterizes the elderly subject is greater constipation leading to the use of laxatives, whose prolonged consumption has harmful effects on the entire

intestinal ecosystem, loss of colon tone and a dangerous condition of habit [56,57]. There can be multiple causes of constipation, including eating disorders and dehydration, as well as pathological conditions, gastrointestinal pathologies, neurological or psychological causes [58].

Evidence suggests that age-related intestinal dysbiosis may contribute to unhealthy aging [59–63]. Since the gut microbiota communicates with the host through various biomolecules, pathways independent of the signalling of nutrients and epigenetic mechanisms, an alteration of these communication pathways related to age-related intestinal dysbiosis can heavily influence the health and life span of the host and trigger an innate immune response [45]. The circulation of bacterial compounds in the host is probably due to the breakdown of the intestinal epithelial barrier caused by the silent chronic inflammation state, and greater intestinal permeability has been suggested as a potential source of age-related inflammation [64–66].

The link between intestinal dysbiosis, chronic inflammation and fragility has been highlighted with intestinal permeability biomarkers [65]. These factors can lead to greater adherence and loss of various microbes and microbial derivatives and increase the host's susceptibility to various local but also systemic disorders through the gut-brain axis, the gut- liver axis, the gut-lung axis [67]. Therefore, in this context, microbial metabolites play an important role in human longevity. However, it is not clear whether the condition of intestinal dysbiosis is a cause or rather a consequence of aging and associated inflammatory disorders. The composition of the gut microbiota is related to circulating cytokine levels and health indicators in the elderly [12]. If the gut microbiota is an age-associated inflammation factor, this would mean that age-related changes in the gut microbiota represent a form of microbial dysbiosis.

A physiological translocation of microbial products is present throughout life; however, with aging, this microbial translocation increases and favours dysbiosis conditions. This feed-forward process increases over the years. In conditions of alteration of the epithelial barrier, the COVID-19 virus finds a fertile ground. Age-associated inflammation is a strong risk factor for mortality in the elderly. Patients with higher levels of inflammatory markers are more likely to be hospitalized, and have higher mortality rates, are fragile, are less independent and are more likely to experience late disease [64,67]. Finally, inflammation in the elderly increases the susceptibility to pneumococcal infection, and is associated with a rise in disease severity and reduced survival [68].

Current research confirms that the intestinal microbiota is significantly altered in SARS-CoV-2 infection, highlighting the crucial role of microbiota in modulating the human response to SARS-CoV-2 infection [25,28]. Interestingly, the alterations are characterized by an opportunistic growth of pathogens while, at the same time, there is a dramatic decrease in beneficial commensal microorganisms [25].

Studies have reported the intimate relationship between infection and gut microbiota dysbiosis and have shown that infection is associated not only with gut bacteria but also with resident viruses. A study reported that treatment with *Lactobacillus brevis* OW38 to aged mice reduced the lipopolysaccharide (LPS) level in colon fluid and blood. Administration of *Lactobacillus brevis* OW38 reduced the ratio of Firmicutes or Proteobacteria to Bacteroidetes. In addition, this lactic acid bacterium was able to inhibit the expression of inflammatory markers, such as myeloperoxidase, TNF, and IL-1β, and inhibited NF-κB activation [69].
