The Origin and Maintenance of Tuberculosis is Explained by Its Subclinical Course, the Neolithic Revolution Being the Trigger for Its Devastating Deadly Drift

(p). The model fits current accepted growth rates for Middle Paleolithic (0.003%/year) and Neolithic (0,1%/year). The data obtained links the origin of M. tuberculosis ancient lineages in the Middle Paleolithic to the induction of mild TB forms (Sputum negative), thanks to a high p factor that was further enhanced by evolution towards modern lineages. The poor health status linked to the unequal society existing after the Neolithic revolution increased the incidence of more severe forms of TB (Sputum positive). This data supports the origin of TB as a well-tolerated highly persistent infection which could coevolve towards mutualism, shows the difficulty of eradicating it and highlights the imperative of providing better health conditions to humans to avoid its severity.

Several models have been set up to try to understand the origin Mtb infection in the Middle Paleolithic, with such a low-density population. One of the initial ones is based on the hypothesis that the mechanism of infection of Mtb was originally based on a late progression towards active disease (i.e. prolonged latency) of more than one generation, with the possibility that younger and more susceptible individuals became infected 28 . Adapting this criteria, Zheng et al. 29 built a model of TB transmission 30,31 using a population of 100 individuals. They concluded that to sustain TB, Mtb would have had to have a high progression to disease of up to 50%, which clearly exceeds the value of 5-10% accepted nowadays 32 .
Recently we developed a SEIR model which showed an extraordinary impact of Mtb, which provoked the extinction of the infected groups 23 . This could only be overcome by an unprecedented population increase attributing an annual population growth rate of 1% and 2.6% instead the accepted 0.003% and 0.1% for the Paleolithic and Neolithic 18 . This data had major drawbacks as it required a dramatic rethink of the population growth parameters in Prehistory.
Recent data based on a precise determination of mortality and curation in the natural history of TB in the pre-chemotherapy era oblige us to modify this model. In this work, the authors have been able to better distinguish the prognostic of sputum positive (SP) and sputum negative (SN) patients, as a sign of TB severity 33 . This data shows a dramatic difference between both forms and allows us to better explore the trade-offs that made this coevolution possible. Severe forms (SP) had an annual mortality ratio of 0.389 and self-curation of 0.250, while mild forms, (SN) have values of 0.025 and 0.125 respectively, thus reducing the mortality by 15 times. Taking the TB disease spectrum into account, especially the impact of mild forms is paramount, as it drastically reduces the mortality but maintains the possibility of disseminating the infection, even when at a lower ratio. It is known that people with SP have ten times higher levels of bacilli in their sputum than SN 34 . Thus, we have built a new SEIR model (TBSpectr) in which we distinguish both clinical forms and allocate them according to a protective factor "p" depending on the health status of the host, which determines the induction of one or the other clinical form.
The main objective of our study has been to evaluate the minimum rate of protection needed to induce mild SN TB in order to maintain the consensual annual population growth established in the Paleolithic and Neolithic societies, and also to determine the impact of ancient and modern Mtb lineages. 4 Our work sustains that Mtb and modern humans could coevolve thanks to the presence of SN lesions, due to the better health status present before the Neolithic Revolution. It was precisely this cultural change that led to the increase of SP forms, even when the predominant Mtb lineages (modern ones) were less virulent. This data supports the extreme conservation of the Mtb virulence factors 35 , generated after a long evolutionary process in mycobacterial species, and which had originally stabilized to generate minimal lesions. The establishment of less equal and more stressful societies after the agricultural revolution means that Mtb starts to have a large impact on health. This is in contrast to what was seen during the Paleolithic when it took advantage of persistence in a mutualist relationship by stimulating the innate immune response (i.e. in form of trained immunity). This data highlights the character of TB as a poverty-related disease, and its higher impact on socially depressed sectors of the population. On the other hand, the benign nature of its origin makes Mtb a highly adapted pathogen, which will be difficult to eradicate using the current diagnostic methods.

2.1.-Mtb infection in the Paleolithic was possible thanks to high protective values.
Adjustment of the continuous TBSpectr model (Figure 1) to the currently accepted human population growth rates in the Paleolithic (0.0003%/year) and Neolithic (0.1%/year) ( Figure 2) gives us the relation between the natality ( ) and the protective factor (p) that determines the allocation towards SN or SP TB forms. We have considered the demographic parameters for each period of time and Mtb lineage (Table 1). Once obtained we established the natality value ( ) that was able to fit for both Mtb lineages in the Paleolithic (0.032) and Neolithic (0.044) periods, considering the lowest and the highest value, respectively. This has allowed us to determine the value for the protective value (p) corresponding to each Mtb lineage for each cultural period. Thus, the protective values were higher in the Paleolithic (0.488), corresponding to the better health status, which was then increased by the irruption of the modern lineages (0.677), which also corresponds to its lower virulence. In the Neolithic period the clinical forms worsened dramatically according to the protective values, which decreased in both modern and ancient lineages to 0.263 and 0.096, respectively.
We have also looked at the number of children of 15 years old or more ( Figure 2B), as the number of fertile individuals available is an interesting factor that contributes to population growth. In this case the difference was not that high between the two periods of time (2.11 vs 2.33) Figure 3 shows the dynamics of the different compartments studied according to the protective factor (p). It is important to note the parabolic evolution vs the exponential decline in SN and SP infectious cases respectively. This indicates the need for the initial Mtb strains, belonging to ancient lineages, to have a higher virulence than the modern ones, otherwise they would contribute to an increase in the "p" value, which would lead to a dramatic reduction of SN lesions, which would probably lead to the clearance of Mtb. Another interesting point is that the percentage of SP infectious cases is not influenced by the Mtb lineage, it depends entirely on the historical period studied, meaning that the reduction of health status in the Neolithic was responsible for the marked increase the severity of TB. The better health status was important in the Paleolithic when we look at the percentage of Recovered and Infectious SN, as it shows a wider gap in the Paleolithic than in the Neolithic. This means that even when ancient lineages were more virulent, because they had originated in the Paleolithic, they were well tolerated. On the other hand, modern lineages increased the percentage of people exposed regardless the historical period studied, confirming its higher capacity for dissemination.

2.3.-The SN Recovered cases were crucial for maintaining Mtb infection in the Paleolithic while the Neolithic era is marked by the entrance of SP lesions.
The evolution of the people in the different compartments over a thousand years using the continuous TBSpectr model to find the stationary distribution ( Figure 4) shows that the SN Recovered compartment was the most important one for ensuring the persistence of Mtb infection. This led to the induction of a reduced percentage of infectious people with SN lesions. The impact of SP infectious cases was minimal in this period. The irruption of modern lineages led to a slight increase on these values, making the SP infectious values even more negligible, but noticeably increasing the number of people exposed, which increased to reach a similar percentage as the group of susceptible people.
This scenario changes radically in the Neolithic period when SP infectious cases irrupt dramatically, as shown in Figure 3. This is remarkable in the case of infections caused by ancient lineages. In the case of the modern ones this irruption is delayed by a parallel increase in SN lesions, although once established, the increase is faster in both cases. In this case the number of SN and SP infectious cases are similar in the stationary equilibrium, which allows a reduction of non-infected people (susceptible compartment). However, in the end it allows a faster growth of the global population, which can be seen by looking at the higher number of people in all compartments after the 1,000-year period and explains the predominance of modern lineages (Supplementary Figure 1).

2.4.-Persistence of Mtb infection required interrelations among hunter-gatherer groups in the Paleolithic while the risk of human extinction was negligible.
When interrogating the discrete TBSpectr model to evaluate the probability of Mtb infection clearance ( Figure 5) it is clear that regardless of the lineage, its survival would be impossible in reduced human groups. Data show how a minimum of a 1,000-person community was necessary to maintain it during the Palaeolithic, and slightly less under the infection with modern lineages. This means that the groups of 50 hunter-gatherers had to interrelate, otherwise the infection would have disappeared. This factor was less important in the Neolithic due to the higher growth population rate. On the other hand, data on the capacity of Mtb infection to cause the extinction of humankind ( Figure 6) shows that this was very low, roughly around 0.2% after 500 years of coevolution in a limited group of 50 people.  Table 2 analyse the influence of the different factors used to define the TBSpectr model. The most important ones are precisely the ones that we have discussed as differentiating ancient and modern Mtb lineages, i.e. the people infected per case/year (e) and rapid progression towards active TB (f), as these are the determinants of induction for all compartments of infected people. In this regard, the immunity factor (i) seems to have the same influence, but in this case has to be read inversely, as the lower the value the higher protection. It was interesting to confirm that the increase in the protective factor (p) increased the number of SN cases and reduced the SP ones, as expected. The reduction of infected cases by the higher mortality caused by TB (µTBsp) was also expected, as it reduces the chances of infection, thus causing an increase in the susceptible compartment. The increase of natality linked to the increase of natural mortality (µ) is also expected as it is precisely what happens in the Neolithic. What is interesting to note is the positive correlation between the curation of SP infectious people (csp) and the number of SN infectious people, showing an obvious interrelation between both compartments. Finally, it is also interesting to see the corroboration of how the charge value (k), which decreases the bacillary load by a factor of 10, increases the chance of SN infectious cases developing from recovered cases. This links the importance of the bacillary load to the induction of SN or SP forms. The higher load should induce SP forms and a lower load SN forms.

3.-DISCUSSION.
Our model demonstrates how coevolution between Mtb and humanity has been possible. Even when molecular measurements point to the origin of the most recent common ancestor (MRCA) of Mtb emerging around 73,000 years BCE, it has been previously hypothesized that its origin is linked with the control of fire, around 300,000 to 400,000 BCE 36 . Furthermore, there is one report that claims the presence of Leptomeningitis tuberculosa in the endocranial surface of a hominid fossil with an estimated age of around 500,000 BCE in Kocabas (Turkey) 37 . Although the interpretation of this finding has been controversial 38 , it could be hypothesized that before a complete settlement as a human parasite, Mycobacterium species have attempted several times to become the MRCA of the Mtb complex that we identify nowadays. In this sense, Gutierrez et al identified a very ancient ancestor (3,000,000 BCE) linked to the smooth tubercle bacilli (M. canettii strains), which are still isolated from human TB today 39 . This position is supported by looking at the evolution of the lipid composition of the mycobacterial cell wall. In this regard Jankute et al have theorized the origin of Mtb as a progressive increase in the hydrophobicity of the cell wall. Thus, the origin of Mtb complex would be M. kansasii, with a smooth morphology; a hydrophilic, environmental mycobacteria which acts as an opportunistic pathogen of several mammals, including humans. With the progressive loss of the polar sugars together with the acquisition of apolar ones, there was an evolution towards M. canettii and canetti/tuberculosis species to finally become the Mtb complex MRCA, and a human obligate pathogen 40 .
Overall this means that Mtb is the result of thousands of years of evolution of environmental mycobacteria, living in cell free media (water and dust) or colonizing free living amoebas 41,42 , moving to colonize the "pulmonary amoebas", as we can consider the alveolar macrophages, to finally becoming an obligate parasite thanks to its capacity to disseminate through aerosols 40 . But originally this parasitization had to be sustainable in the context of a low-density human population; based in small tribal groups with a necessary interrelation between them, as has been demonstrated by others 43 , to avoid the clearance of the infection. Our data indicates that even by developing mild SN TB forms with low dissemination capacity, the obligate parasitization was possible. In fact, recent data on molecular epidemiology supports a higher impact of subclinical TB cases than expected 44 , challenging the status of current diagnosis methodology, if we are to finally eradicate this infection. In fact, looking at the countries with the lowest TB incidence it appears that there is a long-lasting persistent low incidence of the infection 45 , which is usually attributed to imported cases, but that we should also attribute to the original nature of Mtb infection. The persistence of Mtb in our tissues would also provide some evolutive advantage to humans, for instance by increasing trained immunity and allowing them to better control acute respiratory viral infections 46 . A sort of mutualism, the "oldfriends" relationship that has been also linked to several colonizing microorganisms, through the balance of our inflammatory responses, which has led to the evolution of the human immune system 47,48 . Our data also indicates that it was after the change in the living conditions of humans and reduction of standard of living, which came with the Neolithic revolution, that there was an increase in TB severity and mortality. This took place despite the lower virulence of the newly evolved modern lineages, which had a lower inflammatory capacity, but also had higher dissemination abilities 23 .
How could the new Neolithic culture influence the development of more severe SP TB forms? The answer could lie in the neuroendocrine stress response. Evolutionarily speaking this is a very ancient response that can be detected in the first complex multicellular animals (i.e. fish). This reaction, known as the "fight or flight" reaction, increases secretion of glucocorticoids (GCs) 49 . Several studies have demonstrated that lower socioeconomical status is related to stress, and to increased levels of cortisol 50 . It is worth highlighting that with the onset of the agricultural revolution, social imbalance appeared and the transition to an unequal society far from the collaborative one of the Paleolithic 25,26 . Thus, the development of more severe SP TB forms can be attributed to a chronic increase on the levels of cortisol, instead to an increase in Mtb virulence as, on the contrary, modern lineages are less proinflammatory 51 and less prone to induce severe lesions. In a way this coincides with the concept of TB as a disease of the lower classes and a sign of unequal society 52 . There are several observations that supports this hypothesis; from the increased basal cortisol levels recorded in farmers in Kenya, who depend solely on agriculture for their income 53 , or in non-politically influential men of the Bolivian Tsimane forager-horticulturalists 54 . Equally, studies in seven primate species showed that subordinate baboons had higher resting cortisol levels than did their dominant companions 55 .
Glucocorticoids (GC) mobilize neutrophils and generate neutrophilia, increasing their life span by reducing their apoptosis, and increasing IL-1 levels 56 . Equally, oral administration of GC increases neutrophils in airway mucosa due to the increased expression of IL-8 and IFNgamma inducible protein (IP-10) in the epithelia in asthmatic patients [57][58][59] . Furthermore, cortisol increase has a direct impact on the proinflammatory activation of neutrophils, as it also causes hyperglycaemia, which promotes NETosis in lesions 60 . This is in line with the "fightor-flight" paradigm. The acute stress response serves as an endogenous psychophysiological adjuvant that enhances immune responses and has evolved by virtue of the fact that many stressful situations (aggression, accident) result in immune activation (against wounding or infection) 61 . Interestingly, this mechanism would also be the explanation of the sexual dimorphism detected in the TB incidence 62 . Even when there are no differences in basal levels, there are several studies concluding that men produce more circulating levels of cortisol than women in psychological stress tests [63][64][65] . This is the explanation for why major depression increases the risk of TB, but only in men, as observed in a nationwide population study in Korea 66 .
How can we link the increase of GC and severe forms of TB? Recently, a new mechanism has been proposed, which links TB to a sudden inflammatory response led by neutrophils, thanks to the capacity of Neutrophilic Extracellular Traps (NETs) or NETosis for promoting extracellular bacillary growth within themselves [67][68][69] . This leads to the formation of new surrounding granulomas and overall coalescence, in a way resembling soap bubbles, this increases the size of the lesion, generating of a liquefacted necrosis and cavitation 70 .
Our work sustains the hypothesis/idea that Mtb did not only evolve to become a competent aerosol disseminating pathogen among humans, but also the ability to remain sustainable among low density populations. This was possible thanks to its ability to generate minimal lesions (SN forms), and probably because there was some sort of evolutionary advantage to humans in becoming a mutualist microorganism, such as the stimulation of the innate immune response to respond to respiratory viral infections through the trained immune mechanism 71 . It was the cultural human changes in lifestyle, with the Neolithic Revolution, that broke this 8 balance and generated TB the major killer of humankind.
Overall, our data supports the concept that Mtb infection is highly adapted to persist and coevolve with humans. From a practical point of view, this means that its eradication would be very difficult because of its capacity to remain among us discretely, thus challenging the current diagnostic tools. It also emphasizes the poverty-related nature of the disease and the need to provide better global health status and greater equality to avoid severe forms of the disease in order to be able to reduce its terrible morbidity and mortality.

4.1.-TBSpectr model.
We have designed a compartmental mathematical model based on a set of differential equations to describe the dynamics of the evolution of MtbC infection in the population, based on our previous study in several models 23 .
The population is divided into several compartments according to their status with regards to the infection cycle: S, susceptible; E, non-infectious exposed; ISP, smear-positive infectious; ISN, smear-negative infectious; RSP, recovered from a smear-positive TB course; RSN, recovered from a smear-negative TB course. Newborn individuals appear at a rate in the susceptible compartment. Mortality is given by when it is not caused by a TB, $%,'( when it is caused by a SP tuberculosis, and $%,') when it is caused by a SN tuberculosis. Flows between compartments are shown in Figure 1 and given by this set of equations: year post-infection or reinfection is given by f 72,73 . The parameter r provides the risk of disease caused by reinfection as = f + (1 − ) . We consider a protective ratio of 0.1 (i.e., those infected people that do not develop the disease have a protection against the onset of disease of at least 90%) 74 . The drainage of the infection on exposed individuals is given by 75,76 , which is assumed to be reduced by the possibility of endogenous or exogenous reactivation of the infection, defined as and , respectively. Thus, bacillary drainage is defined as: = 0.1 -a -r. Uys et al. 77 determined that recovered subjects have a sevenfold higher chance of developing disease, which is given by w in our model. The birth rate ( ) is determined from the mean number of births per fertile woman/year ( ).
The assumptions behind each of the equations and values have been discussed at length previously 23 . The main novelty of this update of the model is the distinction between smear positive and smear negative infections. In this regard, we have introduced the protection factor p, which determines the percentage of active cases that show a smear negative course. The annual spontaneous cure ( '( and ') ) and dying (μTB,SP and μTB,SN) rates depend on the SP or SN nature of the disease and is given by Ragonnet et al 33 .

4.2.-Assessment of uncertainty and sensitivity in the system.
The uncertainty and sensitivity analysis was of the TBSpectr model was performed as described in 78 . We used a Latin Hipercube Sampling (LHS) technique to generate 1,000 different parameter sets that are representative of the parameter space (sampling-based method). Parameters were explored between the values shown in Table 2. The Partial Rank Correlation Coefficient (PRCC) was computed at each time step for each of the parameters and susceptible, exposed, infected, recovered and total populations, as well as the annual incidence and death rates. We also computed the final PRCC between input parameters and TB clearance and community extinction, using the discrete resolution (see below). This analysis allows for the assessment of the individual effect of each parameter on each outcome, with a linear discount of the effects of the uncertainty on the rest of the parameters.

4.3.-Continuous and discrete resolution of the models.
The continuous TBSpectr model was numerically integrated with Matlab using the Euler method, with an integration step of 1/10 years. As a result of the integration, we obtain the dynamics of each of the model's variable.
As we discussed in our previous paper 23 , the limited size of some of the communities studied suggests the suitability of exploring a discrete resolution of the model, using natural numbers to describe the variables' dynamics. In such discrete resolution, which is also based on the Euler's integration method, we convert each of the flows at each integration step into a natural number using Poisson random distribution. This use of randomness on the flows' rounding makes it possible that two communities with the same initial conditions and model's parameters diverge on their evolution. In particular, there is a chance that a certain community achieves the clearance of the infection, while other communities remain affected by TB or can even be extinct because of the pathogen.                 The origin and maintenance of tuberculosis is explained by its subclinical course, the Neolithic revolution being the trigger for its devastating deadly drift.
Pere-Joan Cardona, Martí Català, Clara Prats.      Relation between the size of the human group and the extinction of humankind. Heatmap of the end values during 1,000 year's evolution on the TBSpectr discrete model using the initial conditions in each compartment found in the equilibrium phase shown previously (Figure 4). Extinction means the disappearance of humankind in the group explored.

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